Method for Screening and Compositions for Treating Cancers

ABSTRACT

The invention relates to compositions and methods for treating proliferative pathologies, in particular cancers. The invention is based on the identification of particular properties of TReP-132 in the control of the cell cycle and in the control of cell proliferation. Said protein behaves as a tumor suppressor and is therefore a preferred target for developing efficient therapeutic strategies. The invention also relates to compositions, uses and methods for identifying compounds which can modulate the activity of said protein, and which can be used for treating different proliferative pathologies, in particular in humans.

The invention relates to compositions and methods for treatingproliferative pathologies, in particular cancers and more particularlyhormone-dependent cancers. The invention is based on the identificationof particular properties of a protein in the control of the cell cycleand in the control of cell proliferation. Said protein, named TReP-132,behaves as a tumor suppressor and is therefore a preferred target fordeveloping efficient therapeutic strategies. The invention also relatesto compositions, uses and methods for identifying compounds which canmodulate the activity of said protein and which can be used for treatingdifferent proliferative pathologies, in particular in humans.

The functional characterization of the cytochrome P450scc gene promoterled to the isolation of the TReP-132 protein (Transcriptional RegulatingProtein of 132 kDa). Said protein is involved in regulating theexpression of steroidogenic genes and in particular in activating theexpression of the gene coding for the P450scc enzyme (Gizard et al.,2001 and Gizard et al., 2004). Cytochrome P450scc is a mitochondrialenzyme encoded by the CYP11 A1 gene and expressed in steroidogenictissues such as ovary, testis, placenta or adrenal glands (Mellon etal., 1993 and Stromstedt et al., 1995). P450scc catalyzes the firstenzymatic step in steroid synthesis by converting cholesterol topregnenolone (Miller, 1988).

Overexpression of TReP-132 in human adrenal NCI-H295 cells has beenshown to result in a significant increase in pregnenolone production,which is in agreement with the ability of said factor to increaseP450scc gene expression and potentially increase the production ofsteroid hormones, including mineral corticoids, glucocorticoids and sexsteroids.

TReP-132 therefore appears to be involved in multiple physiologicaleffects of steroid hormones.

TReP-132 cDNA (3600 nucleotides) was first isolated by screening a humanplacenta cDNA library. The protein sequence displays the characteristicmotifs of a transcriptional regulatory factor, such as three zinc fingermotifs of the C₂H₂ subtype, regions rich in acidic amino acids, prolineand glutamine, and two LXXLL motifs. Northern blot and RNase protectionassays showed that TReP-132 is expressed in different tissues, withparticularly high expression in steroid target tissues such as uterus,prostate, testis and certain areas of the brain (Gizard et al., 2001;Gizard et al., 2002b and Duguay et al., 2003) as well as in the cancercell lines LNCaP, MCF-7 and T47-D (Gizard et al., 2001).Immunocytochemical studies localized the TReP-132 protein to thenucleus. Based on the primary structure analysis, it appears thatTReP-132 contains several protein interaction domains such as two LXXLLmotifs. Studies in NCI-H295 cells showed that the regulation of geneexpression by TReP-132 involves the formation of a transcriptionactivation complex and a direct interaction of TReP-132 with SF-1(Steroidogenic Factor-1) and the cofactor CBP/p300 (Gizard et al., 2002band Gizard et al., 2004). The TReP-132/SF-1 interaction requires theLXXLL motif located in the N-terminal region of TReP-132 as well as theproximal activation domain and the AF-2 motif of the SF-1 protein.

The interaction of TReP-132 with DNA, the nuclear localization thereofand the involvement thereof in the regulation of gene expressionindicate that TReP-132 is a transcriptional regulatory factor.

The inventors have found that TReP-32 is expressed in different tissues,including non-steroidogenic tissues such as brain, thymus and heart,which, considering its ability to interact with DNA to regulate geneexpression, seems to indicate that TReP-132 may be involved in differentfunctions, other than the regulation of steroid synthesis. Accordingly,the application now demonstrates the involvement of TReP-132 in cellproliferation and associated mechanisms, such as cell differentiationand tissue development. In particular, the application demonstrates aregulation of TReP-132 expression during the cell cycle, and reveals arole of said factor in regulating cell proliferation.

More particularly, the inventors have shown that TReP-132 is involved incell proliferation dependent on steroid hormones or analogs thereof andmore specifically progesterone, particularly in cancers affectingsteroid hormone-responsive organs and typically female cancers, inparticular breast and uterine cancer. In fact, steroid hormones(estrogen and progesterone) play a key role in mammary glandproliferation and differentiation (Musgrove et al., 1994). In vitrostudies in T47-D cell lines have shown that the cellular response toprogesterone or its derivatives R5020 and ORG 2058 (Stahlberg et al.,2003) is biphasic: initial exposure leads to proliferation whereasprolonged exposure results in inhibition of cell growth. Thus, long-termtherapy with synthetic progestins such as megestrol acetate ormedroxy-progesterone acetate is used clinically in the treatment ofcertain breast cancers (Sitruk-Ware et al., 1999 and Ingle, 2002).

The application demonstrates the role of TReP-132 as a regulator of cellproliferation. The inventors have shown that transfection of HeLa cells,which do not endogenously express PR (Progesterone Receptor), with aTReP-132 expression vector leads to fewer colonies than transfectionwith an empty vector alone. This finding indicates that TReP-132expression decreases or arrests cell cycle progression. Furthermore,cells expressing exogenous TReP-132 have a significantly longer doublingtime than control cells and the colonies obtained after TReP-132transfection are smaller.

In parallel, the inventors have shown that TReP-132 expression isincreased in T47-D cells after treatment with progesterone and thatselective inhibition of TReP-132 increases cell proliferation andabolishes the inhibitory effect of progesterone. These findingsdemonstrate the inhibitory effect of TReP-132 on progesterone-dependentcell proliferation. The involvement of TReP-132 in cell proliferationwas subsequently demonstrated in human breast cancer MCF-7 epithelialcells: treatment of said cells with estradiol led to a significantdecrease in TReP-132 expression and a concomitant increase in cellproliferation. These findings demonstrate the inhibitory effect ofTReP-132 on progesterone-dependent and progesterone-independent cellproliferation.

In addition, the application demonstrates a regulation of TReP-132expression during the cell cycle. It was shown that in the absence ofestradiol treatment, a higher proportion of MCF-7 cells were in G₁phase, thereby suggesting that TReP-132 expression arrests cell cycleprogression from G₁ to S phase.

The involvement of TReP-132 in the G₁ phase of the cell cycle wasfurther demonstrated by the finding that said protein was expressedexclusively during the G₁ phase of the cell cycle followingsynchronization of HeLa cells.

In addition, the inventors have shown that overexpression of TReP-132 inT47-D cells induced an arrest of cell proliferation in the G₁ phasetogether with an inhibition of CDK (Cyclin Dependent Kinase) activityand a decrease in pRb phosphorylation (Retinoblastoma protein). Thesefindings reveal a negative correlation between TReP-132 expression andcell cycle progression.

The effects of TReP-132 overexpression on the level of cyclin-dependentkinase inhibitors (CDKIs) have also been determined. In so far asTReP-132 has been shown to be involved in regulating gene expression, itwas of interest to identify target genes regulated by TReP-132 which areinvolved in the cell cycle. RNase protection and reporter gene assaysshowed that induction of TReP-132 expression is associated with anincreased expression of the CDKIs p21, p16 and p27. The inventors havealso performed transfection, immunoprecipitation and gel shift studiesshowing that TReP-132 activates the p21, p16 and p27 gene promotersthrough a mechanism involving a complex formed by TReP-132, the Sp1transcription factor and PR. These data therefore indicate that TReP-132acts as a cofactor of PR (Progesterone Receptor), in the regulatorycomplex formed with Sp1.

Likewise, the inventors have found an interaction between TReP-132 andthe estrogen receptor (ER).

The inventors further suggest that TReP-132 expression is associatedwith a low tumor incidence and low tumor aggressiveness. Interestingly,it has been shown that chromosome 6, which carries the TReP-132 gene inposition 6p21.1-p12.1 (Gizard et al., 2002a), is the fourth mostfrequently rearranged chromosome in human tumors and that the 6p21 locusis a hot spot for mutations associated with immortalizing events in somecancers. Mutations altering the expression or activity of TReP-132 mighttherefore be associated with tumor development. TReP-132 might thereforebe a marker of predisposition, cell differentiation and development, andtumor aggressiveness.

The application also demonstrates the role of TReP-132 in celldifferentiation. Gizard et al. (2004) suggest that TReP-132overexpression in human adrenal NCI-H295 cells induces thedifferentiation of zonae fasciculata and reticularis cells and thatTReP-132 induces the expression of the P450 aromatase gene required formammary gland differentiation. Moreover, inhibition of T47-D cellproliferation by progesterone, which the inventors have shown increasesTReP-132 expression, is related to the induction of differentiationprograms (Musgrove et al., 1998). This hypothesis was confirmed by theup-regulation of p21, p16 and p27 which are molecules that facilitatehormone-dependent differentiation in many cell differentiation pathways(Inoue et al., 1999). In breast tissue, p27 up-regulation underlies thearrest of cell differentiation at the alveolar phase (Said et al., 2001)and high levels of p21 and p27 are respectively associated withintermediate and advanced states of differentiation in ductal carcinomain situ (DCIS—a non-invasive form of breast cancer) (Mommers et al.,2001). Considering that TReP-132 and progesterone have similar effectson the expression of p21, p27 and p16, it clearly appears that TReP-132is a marker of cell differentiation, in particular of breast epithelialcells, and that TReP-132 is a differentiation factor.

The activities of TReP-132 so demonstrated indicate that the TReP-132gene is potentially a tumor suppressor gene. As shown for other tumorsuppressor genes such as p53 which also activates cyclin-dependentkinase inhibitor genes like p21, TReP-132 probably also regulates thecell cycle through many growth inhibition pathways. This includespositive transcriptional regulation of target genes by promotingapoptosis and of growth inhibitor genes, as well as negative regulationof other genes, including survival factors and anti-apoptosis genes.

Taken together, these data show that the TReP-132 protein can regulatecell cycle progression in different cell types. TReP-132 is therefore apotential tumor suppressor gene. The regulated expression of TReP-132and the production of functional TReP-132 protein are critical stepsrequired for proper cell cycle progression and constitute particularlyadvantageous targets for therapeutic intervention. These data also showthat compounds which can increase the activity of the TReP-132 proteinpotentially represent potent and selective inhibitors of the pathwaysinvolved in cell cycle progression, uncontrolled cell proliferation,tissue development and/or cell differentiation.

A first object of the invention is based on the use of a compoundincreasing or mimicking the activity of the TReP-132 protein forpreparing a composition intended for the treatment of a proliferativedisease.

Another object of the invention relates to the use of said compositionin combination with a hormone for a simultaneous, separate or sequentialadministration.

The invention also relates to methods for treating proliferativepathologies, comprising administering to a subject a compound increasingor mimicking the activity of the TReP-132 protein, in particular acompound which mimics the activity of the TReP-132 protein, stimulatesthe expression, maturation or nuclear targeting of the TReP-132 proteinand/or increases the concentration of the TReP-132 protein in cells. Theadministration can be carried out by any classical route for this typeof therapeutic approach, such as in particular the systemic route, inparticular, by injection, particularly intravenous, intradermal,intratumoral, subcutaneous, intraperitoneal, intramuscular,intra-arterial, etc.

A particular object of the invention is based on the use of the TReP-132protein or an analog for preparing a composition intended for thetreatment of a proliferative disease.

Another object of the invention relates to the use of a nucleic acidcoding for the TReP-132 protein for preparing a composition intended forthe treatment of a proliferative disease.

Another particular object of the invention relates to a pharmaceuticalcomposition comprising a TReP-132 protein or a nucleic acid coding forthe TReP-132 protein, and a pharmaceutically acceptable excipient. Theinvention also relates to a pharmaceutical composition comprising, in apharmaceutically acceptable excipient, a compound selected in the groupconsisting of the TReP-132 protein, an analog of same, a compoundincreasing or mimicking the activity of TReP-132 and a compoundidentified with the aid of a method according to the invention, combinedwith one or more hormones, in view of a simultaneous, separate orsequential administration in order to treat a proliferative disease.

In a preferred embodiment, the pharmaceutical composition comprises, ina pharmaceutically acceptable excipient, the TReP-132 protein.

In another preferred embodiment, the pharmaceutical compositioncomprises, in a pharmaceutically acceptable excipient, progesterone oran estrogen.

The invention can be used for the treatment of various proliferativepathologies, and in particular for the treatment of cancers. Theinvention is particularly adapted to the treatment of hormone-dependentcancers, in particular cancers which are sensitive to a deregulation inthe synthesis and/or activity of estrogens or progesterone. Inparticular, the invention can be used for the treatment of breast oruterine cancer.

Another object of the application relates to a method for the selection,identification, characterization or optimization of active compoundsmodulating, preferably reducing, cell proliferation comprisingdetermining the ability of a test compound to mimic, increase or promotethe binding of the TReP-132 protein or of a complex comprising theTReP-132 protein to the receptor of one or more steroid hormones or tomodulate the activity of a target gene of said receptor, in particularthe p27, p21 or p16 gene.

The invention further relates to a method for the selection,identification, characterization or optimization of active compounds,comprising determining the ability of a test compound to modulate thebinding of the TReP-132 protein or of a complex comprising the TReP-132protein to the receptor of one or more steroid hormones. Said method forthe selection, identification, characterization or optimization, invitro or ex vivo, of active compounds modulating cell proliferation,comprises:

-   a) contacting a test compound with:    -   i) the TReP-132 protein or a complex comprising the TReP-132        protein, and    -   ii) the receptor of one or more steroid hormones, and-   b) measuring the binding of the TReP-132 protein or of the complex    comprising the TReP-132 protein to said receptor.

In the context of the invention, the receptor of one or more steroidhormones is in particular the progesterone receptor (PR) or the estrogenreceptor (ER).

In particular, the invention relates to a test compound which increasesor promotes the binding of the TReP-132 protein or of a complexcomprising the TReP-132 protein to the receptor of one or more steroidhormones (for example the progesterone receptor), or else a testcompound which reduces or inhibits the binding of the TReP-132 proteinor of a complex comprising the TReP-132 protein to the receptor of oneor more steroid hormones (for example the estrogen receptor).

Another object of the invention is based on a pharmaceutical compositioncomprising, in a pharmaceutically acceptable excipient, a compoundmodulating the binding of the TReP-132 protein to the receptor of one ormore steroid hormones.

The application also relates to a method for the selection,identification, characterization or optimization, in vitro or ex vivo,of active compounds modulating, preferably reducing, cell proliferation,comprising:

a) contacting, in the presence of progesterone or estrogens, a testcompound with:

-   -   i) respectively, the progesterone or estrogen receptor,    -   ii) Sp1, and    -   iii) a nucleic acid comprising all or part of the promoter of a        target gene of progesterone or estrogens, and        b) measuring the binding of Sp1 or of the complex comprising the        progesterone or estrogen receptor and Sp1 to said promoter, or        measuring the expression of said promoter, said measurement        being an indication of the effect of the test compound on cell        proliferation.

According to another embodiment of the invention, the method is carriedout in the presence of the TReP-132 protein or a complex comprising theTReP-132 protein.

In a preferred manner, said method can be carried out in a cell.

In the context of the invention, the target genes of progesterone orestrogens are the p21, p16 and/or p27 genes.

Another aspect of the invention relates to a method for detecting apredisposition, diagnosing, monitoring and measuring the aggressivenessof a proliferative disease comprising detecting, in a patient, theoverexpression of estradiol, the reduction in progesterone expression ora decrease in the expression of the CDKIs p21, p16 and/or p27.

As indicated, the invention is based on the demonstration that theTReP-132 protein can act as a cell cycle regulator, and in particularcan inhibit the proliferation of cancer cells, in particular inhormone-dependent cancers.

In the context of the invention, the pharmaceutical compositionscomprise in particular compounds increasing the binding of the TReP-132protein to the receptor of one or more steroid hormones, in apharmaceutically acceptable excipient.

The invention also relates to the use of a compound increasing,promoting or facilitating the binding of the TReP-132 protein to thereceptor of one or more steroid hormones for preparing a compositionintended for the treatment a proliferative disease.

Another particular object of the invention relates to a method forpreparing a pharmaceutical composition for treating a proliferativedisease comprising a step which employs a method such as describedherienabove.

A particular object of the invention relates to the treatment ofdifferent proliferative pathologies, such as cancer or stenosis,advantageously cancer. In particular, the invention can be used in thetreatment of hormone-dependent cancers, and in particular in thetreatment of breast cancer or uterine cancer.

In the context of the invention, the receptor of one or more steroidhormones is in particular a progesterone receptor or an estrogenreceptor.

TReP-132 Protein

In the spirit of the invention, the expression “TReP-132 protein”denotes a polypeptide comprising the sequence SEQ ID NO: 2, the naturalor functional variants, derivatives or homologs thereof. Moreparticularly, it is any natural variant of sequence SEQ ID NO: 2,resulting from a polymorphism, splicing, mutation, etc. Said naturalvariants can therefore comprise one or more modifications such assubstitutions, insertions and/or deletions of one or more residues, etc.The term homolog should be understood to mean TReP-132 proteins fromother species, for example rodents, bovines, etc. Preferably, it is apolypeptide recognized by a polyclonal antibody produced from theTReP-132 protein corresponding to sequence SEQ ID NO: 2.

In general, the term TReP-132 protein denotes a polypeptide havingsequence SEQ ID NO: 2.

Preferred variants contain at least 80% of sequence SEQ ID NO: 2, morepreferably at least 90% identity with sequence SEQ ID NO: 2. The degreeof identify can be determined by example by using the CLUSTAL method.Particular variants contain a mutation or substitution affecting at mostfive amino acids of sequence SEQ ID NO: 2. In the spirit of theinvention, particular functional variants are variants resulting fromsplicing event(s), for example the variants described in GenBank underthe reference numbers AJ277276 and AJ277275.

Particular functional variants according to the invention are fragmentsof the TReP-132 protein described hereinabove, in particularpolypeptides comprising part of the sequence SEQ ID NO: 2. Thepolypeptide fragments of the invention preferably contain fewer than 200amino acids, more preferably fewer than 150 amino acids. A TReP-132protein according to the invention can also comprise heterologousresidues, added to the indicated amino acid sequence. Thus, one objectof the invention is a polypeptide comprising all or part of sequence SEQID NO: 2 or a natural variant thereof and a heterologous part. Theheterologous part can correspond to amino acids, lipids, sugars, etc. Itcan also be chemical, enzymatic, radioactive group(s), etc. Inparticular, the heterologous part can be a marker, targeting agent,stabilizer, agent facilitating production, a protective agent, an agentfacilitating entry of the protein into cells, a toxin, an activecompound, an antibody, etc.

In a general manner, the expression “TReP-132 protein” designates apolypeptide corresponding to SEQ ID NO: 2 or any polypeptide coded by anucleic acid which hybridizes with sequence SEQ ID NO: 1 or with aregion thereof (typically comprising at least 50 bases), in conditionsof moderate or high stringency. Suitable stringency conditions are forexample an incubation at 42° C. for 12 hours in a solution comprising50% formamide, 5×SSPE, 5×Denhardt's, 0.1% SDS, (1×SSPE is composed of0.15 M NaCl, 10 mM NaH₂PO₄, 1.3 mM EDTA, pH 7.4). Of course it ispossible to vary the temperature and salt concentration of the medium.

In the context of the invention, the term TReP-132 protein analogdenotes any functional analog of the TReP-132 protein. Said functionalanalogs are polypeptides, optionally comprising a heterologous part suchas defined hereinabove, displaying at least one biological property ofthe TReP-132 protein, in particular the ability to bind a protein,preferably a hormone receptor and in particular a progesterone receptoror an estrogen receptor, or else the ability to regulate the expressionof the p27, p21 and p16 genes. In particular, a TReP-132 protein orvariant according to the invention comprises at least one LXXLL motif.

In the spirit of the invention, the term “complex comprising theTReP-132 protein” denotes any type of complex in which the TReP-132protein is involved. Said complex can comprise many partners, such asnuclear receptors, cofactors, and the like, which can have differentnatures and functions. Cofactors can be exemplified by transcriptionfactors, in particular p300, Sp1 or VDR (Vitamin D Receptor). Receptorsare in particular nuclear receptors among which one can mention thereceptors of one or more steroid hormones, orphan receptors, the PPAR,LXR, RXR, FXR receptors or any other nuclear receptor. In a preferredmanner, the receptors according to the invention are involved in cellproliferation or differentiation. More preferably, the receptors of oneor more steroid hormones are the progesterone receptor (PR) and theestrogen receptor (ER).

A TReP-132 protein according to the invention can be prepared by anymethod known to those skilled in the art, in particular by artificialsynthesis and more particularly by solid phase synthesis, or by anybiological, genetic or enzymatic method, and in particular by expressionof a nucleic acid encoding said protein in a suitable host cell. In thecontext of the invention, a preferred TReP-132 protein is a humanTreP-132 protein having a sequence identical to sequence SEQ ID NO: 2for example.

Compound

An object of the invention is based on a method for the selection,identification, characterization or optimization of active compoundsmodulating, preferably reducing, cell proliferation, comprisingdetermining the ability of a test compound to mimic, increase or promotethe binding of the TReP-132 protein or of a complex comprising theTReP-132 protein to the receptor of one or more steroid hormones.

Said method can comprise contacting a test compound with the TReP-132protein or with a complex comprising the TReP-132 protein, and with areceptor of one or more steroid hormones; and measuring the binding ofthe TReP-132 protein or of the complex comprising the TReP-132 proteinto the receptor of one or more steroid hormones.

In the spirit of the invention, the expression “compound increasing,promoting or facilitating the binding of the TReP-132 protein to thereceptor of one or more hormones” denotes any compound, agent, factor,condition or treatment which can increase or stimulate, in a cell, thebinding of the TReP-132 protein to the hormone receptor of interest. Itcan be a ligand of a nuclear receptor, preferably of steroid hormones(in particular protesterone or estrogen) or a steroid analog.

Another object of the invention is based on the use of a compoundmodulating the binding of the TReP-132 protein to the receptor of one ormore steroid hormones, in particular to a progesterone receptor or to anestrogen receptor, for preparing a composition intended for thetreatment of a proliferative disease. According to the invention, themodulation of binding can consist in an increase or a decrease ofbinding of the TReP-132 protein to the receptor of one or more steroidhormones such as those indicated earlier.

In a preferred manner, the invention relates to the use of a compoundincreasing or promoting the binding of the TReP-132 protein or of thecomplex comprising said protein to the progesterone receptor and/ordecreasing the binding of the TReP-132 protein or of the complexcomprising said protein to the estrogen receptor, for preparing acomposition intended for the treatment of a proliferative disease and inparticular a hormone-dependent cancer.

Another object of the invention is based on the use of a compoundincreasing or mimicking the activity of the TReP-132 protein forpreparing a composition intended for the treatment of a proliferativedisease. In particular, the invention can be used in the treatment ofhormone-dependent cancers, in particular in the treatment of cancerssensitive to a deregulation of the synthesis and/or activity ofprogesterone or estrogens such as breast or uterine cancer.

In a preferred embodiment of the invention, the composition is used incombination with a hormone for a simultaneous, separate or sequentialadministration.

In the spirit of the invention, the expression “compound increasing ormimicking the activity of the TReP-132 protein” denotes any compound,agent, factor, condition or treatment which can increase, mimic orstimulate, in a cell, the activity of the TReP-132 protein.

Preferably they are substances, selected for example in the groupconsisting of a nucleic acid, a polypeptide, a lipid, a small molecule,and the like. Said compound can be identified with the aid of a methodaccording to the invention.

The compound which is used is preferably a compound which mimics theactivity of the TReP-132 protein, stimulates the expression or nucleartransport of the TReP-132 protein and/or increases the concentration ofthe TReP-132 protein in cells.

Particularly preferred compounds are those which can selectivelyincrease the activity of the TReP-132 protein, that is to say,essentially without directly and significantly affecting the activity ofanother metabolic pathway.

More specifically, in the spirit of the invention, the term “activity ofthe TReP-132 protein” refers in particular to the synthesis of saidprotein (transcription, translation, etc.), maturation thereof,transport thereof to the nucleus, interaction thereof with a receptor orwith a nucleic acid, the transcriptional activity thereof, thedegradation thereof, etc. The compound increasing the activity of theTReP-132 protein can therefore be an agent increasing the synthesis ofthe TReP-132 protein, an agent increasing the transport thereof, anagonist of the TReP-132 protein, an agent mimicking the activity of theTReP-132 protein, etc.

In a particular embodiment of the invention, a compound which canincrease the synthesis of the TReP-132 protein, that is to say, inparticular the transcription or translation of its gene or RNA, is used.Advantageously, it is possible to use a compound which increases theexpression of the TReP-132 protein in a cell. One object of theinvention is therefore based on the use of a compound increasing theexpression of the TReP-132 protein for preparing a composition intendedfor the treatment of proliferative diseases.

Said compound is preferably a nucleic acid coding for a TReP-132protein, a compound stimulating the promoter of the TReP-132 gene or acompound increasing the stability of TReP-132 mRNA.

In this regard, a particular object of the invention is based on the useof a nucleic acid coding for a TReP-132 protein for preparing a compoundintended for the treatment of a proliferative disease. Another object ofthe invention is based on a pharmaceutical composition comprising anucleic acid coding for the TReP-132 protein and a pharmaceuticallyacceptable excipient.

In the spirit of the invention, the nucleic acid can be a DNA or RNA,for example a genomic, complementary or synthetic DNA, a messenger RNA,etc. Preferably it is a cDNA. A particular nucleic acid codes for aprotein corresponding to sequence SEQ ID NO: 2 or for a natural orfunctional variant of same. More particularly it is any nucleic acidcoding for a TReP-132 protein and which can hybridize with sequence SEQID NO: 1 or with a region thereof (typically comprising at least 50bases), in conditions of moderate or high stringency. Suitablestringency conditions are for example an incubation at 42° C. for 12hours in a solution comprising 50% formamide, 5×SSPE, 5×Denhardt's, 0.1%SDS, (1×SSPE is composed of 0.15 M NaCl, 10 mM NaH₂PO₄, 1.3 mM EDTA, pH7.4). Of course it is possible to vary the temperature and saltconcentration of the medium. In the context of the invention, thenucleic acid can optionally comprise regulatory regions, in particular,a promoter region, poly A, etc. In particular it is possible to usehomologous or heterologous promoters, strong or weak, regulated,constitutive or inducible, tissue-specific or ubiquitous, etc. They canbe of different origins, such as viral, cellular, bacterial, artificialpromoters, etc. Specific examples of promoters include in particular theHSV-LTR, CMV, TK, SV40, PGK, albumin promoter, etc.

The nucleic acid can be cloned in a vector, in particular an expressionvector, such as for example a plasmid, cosmid, phage, virus, artificialchromosome, etc. Preferably it is a plasmid vector or a vector derivedfrom a virus, such as for example a retrovirus, adenovirus, AAV, herpesvirus, etc. Particularly useful viruses are retroviruses, adenovirusesor AAV, whose genome has been modified to contain a region coding for aTReP-132 protein, and so as to be replication-defective. The methods forproducing such defective viruses are known to those skilled in the art,and comprise, for example, introducing a recombinant viral vector in apackaging cell, optionally in the presence of a helper virus or plasmid.The recombinant viruses used in the context of the invention areadvantageously replication-defective, which is to say that they areessentially incapable of autonomously replicating in a cell. Saidrecombinant viruses therefore have a recombinant genome in which one ormore viral genes (or viral regions) essential for replication have beendisabled (e.g., mutated or deleted, in whole or in part), such as forexample the E1 or E4 regions (for adenoviruses), Rep or Cap (for MVs),GAG and/or POL (for retroviruses), etc. Methods for producingrecombinant retroviruses are described for example in WO90/02806, U.S.Pat. No. 5,324,645 and WO94/19478. Methods for producing recombinantadenoviruses are described for example in WO95/02697 and WO96/22378.

The nucleic acid or vector can be used in the context of the inventionto increase the expression and therefore the activity of the TReP-132protein in vitro, ex vivo or directly in vivo. More generally it is adirect use in vivo, in the proliferative cells, or ex vivo, from asample or biopsy of the tissue to be treated, which is then reinjectedinto the patient, typically after irradiation.

Another particular object of the invention is based on the use of acompound stimulating the TReP-132 gene promoter or increasing thestability of TReP-132 mRNA for preparing a composition intended for thetreatment of a proliferative disease. Said compound in fact makes itpossible to increase the amount of TReP-132 protein in a cell.

Another object of the invention is based on a pharmaceuticalcomposition, characterized in that it comprises at least one compoundstimulating the activity of the TReP-132 gene promoter and apharmaceutically acceptable excipient. Said compound can be identified,validated or optimized for example with the aid of a method comprisingdetermining the ability of a test compound to bind to the TReP-132 genepromoter or a region thereof, and/or to activate said promoter. Saidactivity can be determined by measuring the expression of a gene placedunder the control of the TReP-132 promoter.

According to another preferred embodiment, the invention makes use ofone or more compounds which can increase, induce or stimulate thematuration, stability or activity of the TReP-132 protein in a cell. Anobject of the invention is therefore based on a compound increasing thematuration, stability or activity of the TReP-132 protein for preparinga composition intended for the treatment of a proliferative disease. TheTReP-132 protein and analogs thereof are examples of compounds which canbe used.

In fact, according to a preferred embodiment of the invention, it ispossible to directly use the TReP-132 protein, typically produced by invitro recombination, so as to increase the antiproliferative activity.The TReP-132 protein can be produced in vitro by expressing arecombinant nucleic acid in any suitable cell system, and recovering theprotein produced. In particular the cell system is a bacteria (e.g., E.coli), yeast (e.g., Saccharomyces, Kluyveromyces), a eukaryotic cell,particularly human (fibroblast, hepatocyte, etc.).

According to another preferred embodiment of the invention, the compoundwhich can increase the transcriptional activity of the TReP-132 proteinis represented by any compound increasing the binding of the TReP-132protein or of a complex comprising the TReP-132 protein to the promoterof a target gene of the TReP-132 protein. Target gene of the TReP-132protein should be understood to mean any gene whose transcription isregulated by the TReP-132 protein, in particular the p27, p21 and p16genes. In fact, the application shows that the TReP-132 protein canstimulate the expression of the p27, p21 and p16 genes. The inventiontherefore discloses a novel pathway and novel molecular targets, whichcan be used in the scope of efficient therapeutic approaches.

An object of the invention is therefore based on the use of a compoundincreasing the binding of the TReP-132 protein or of a complexcomprising the TReP-132 protein to the promoter of a target gene of theTReP-132 protein for preparing a pharmaceutical composition intended forthe treatment of a proliferative disease.

In a preferred embodiment of the invention, a compound is used whichincreases the activity of the promoter of a target gene of the TReP-132protein.

In the context of the invention, the target genes of the TReP-132protein are in particular the genes coding for p21, p27 or p16. Saidcompound can be selected, validated or optimized by differentapproaches, and in particular by means of a binding assay or atranscriptional assay employing a reporter gene placed under the controlof a promoter having all or part of the sequence of the p27, p21 or p16gene promoter. Said compound can be a peptide derived from the TReP-132protein, an antibody, a small molecule, etc.

While any approach allowing to increase or mimic the activity of theTReP-132 protein can be used in the scope of the invention, moreparticularly preferred are strategies, molecules and conditions allowingto increase the synthesis of the TReP-132 protein, in particular the useof a nucleic acid coding for the TReP-132 protein. In this regard, aparticular object of the invention is based on the use of a nucleic acidcoding for the TReP-132 protein for preparing a composition intended forthe treatment of a proliferative disease.

Another particular object of the invention is based on the use of acompound modulating the formation or activity of the complex comprisingthe TReP-132 protein, Sp1 and a receptor of one or more steroidhormones, e.g., a progesterone receptor or an estrogen receptor, forpreparing a composition intended for the treatment of a proliferativedisease, in particular a hormone-dependent cancer.

Preferably, the test compound modulates the interaction of the TReP-132protein, Sp1 and a receptor of one or more hormones (in particularsteroids, e.g., a progesterone receptor or an estrogen receptor),modulates the binding of the complex comprising the TReP-132 protein,Sp1 and said receptor to the promoters of TReP-132 target genes ormodulates the action of the complex comprising the TReP-132 protein, Sp1and a receptor such as described hereinabove on the promoter of a targetgene. In the context of the invention, the target genes are inparticular the p21, p27 and/or p16 genes.

The invention can be used in the treatment of a proliferative disease,such as cancer. Preferably, the invention is used for treatinghormone-dependent cancers, in particular cancers sensitive to aderegulation of the synthesis and/or activity of progesterone orestradiol, even more preferably, breast or uterine cancer.

Use of the Compounds

Considering the physiological functional properties of the target used,the inventive compounds can be used in the treatment of a proliferativedisease and in particular in the treatment of a cancer. In particular,the compounds according to the invention are compounds which can mimicor increase the activity of the TReP-132 protein, compounds which canmodulate the binding of the TReP-132 protein or of a complex comprisingthe TReP-132 protein to the receptor of one or more steroid hormones,e.g., a progesterone receptor or an estrogen receptor, or compoundswhich can modulate the formation or activity of the complex comprisingthe TReP-132 protein, Sp1 and the receptor of one or more steroidhormones, e.g., a progesterone or an estrogen receptor. In particular,the inventive compounds are compounds identified with the aid of amethod such as described hereinabove.

In the spirit of the invention, the term “treatment” refers topreventive, curative, palliative treatment, as well as management ofpatients (alleviating suffering, prolonging survival, improving qualityof life, slowing disease progression, reducing tumor size), etc.Furthermore, the treatment can be carried out in combination with otheragents or treatments, in particular addressing different metabolicpathways. It can also be combined with hormonal therapy, chemotherapy orradiotherapy, for example. A particular combined treatment comprises thesequential, simultaneous or separate use of a compound increasing ormimicking the activity of the TReP-132 protein and a hormone. Anotherparticular combined treatment involves the sequential, simultaneous orseparate use of a compound stimulating the binding of the TReP-132protein or of a complex comprising the TReP-132 protein to the receptorof one or more steroid hormones, e.g., to the progesterone receptor orto the estrogen receptor, and a hormone.

A particular object of the invention thus relates to the use of acomposition according to the invention for treating a proliferativedisease in combination with a hormone for a simultaneous, separate orsequential administration.

The invention can be used for the treatment of proliferative diseasesaffecting mammals, and humans in particular. The data presented in theexamples demonstrate the ability of the TReP-132 protein to inhibit cellproliferation, in particular that of tumor cells, and to block cellcycle progression.

In the spirit of the invention, proliferative disease is understood tomean any pathology characterized by or associated with cell cyclederegulation and/or uncontrolled or pathological cell proliferation.Typical examples of such pathologies are in particular cancers,stenosis, fibrosis, psoriasis, etc. The invention is particularlyadapted to the treatment of cancer, and in particular solid tumors,primary or metastasized. In particular these include cancers of thelung, liver, colon, head-and-neck, brain, bladder, spleen, skin, breast,prostate, uterus, thymus, etc. In particular they are hormone-dependentcancers and in particular typically female cancers represented by breastand uterine cancer.

An object of the invention is based on the use of a compound such asdefined hereinabove for preparing a composition intended for thetreatment of a proliferative disease, in particular for the treatment ofa cancer. The invention also discloses methods for treatingproliferative diseases based on the use of the aforementioned compounds.

The invention relates in particular to the use of a compound such asdefined hereinabove for reducing the proliferation of tumor cells in apatient, and to a corresponding method.

The invention also relates to the use of a compound such as definedhereinabove for reducing tumor progression, or for inducing shrinkage ofa tumor in a patient, and to a corresponding method.

The invention further relates to the use of a compound such as definedhereinabove for inducing apoptosis of tumor cells in a patient, and to acorresponding method

Screening Tests

The invention relates to methods for the selection, identification,characterization or optimization of active compounds modulating,preferably reducing, cell proliferation, based on measuring themodulation of binding or activity of the TReP-132 protein or of acomplex comprising the TReP-132 protein to a hormone receptor or targetgene, measuring the modulation of binding or activity of the complexcomprising the TReP-132 protein and a hormone receptor on the promoterof a target gene or measuring the modulation of binding of the complexcomprising the TReP-132 protein, a hormone receptor (e.g., aprogesterone or estrogen receptor) and its hormone and the Sp1transcription factor to the promoter of a target gene.

In particular the invention relates to methods for the selection,identification, characterization or optimization of active compoundsmodulating cell proliferation, comprising determining the ability of atest compound to modulate, i.e. mimic, increase or promote the bindingof the TReP-132 protein or of a complex comprising the TReP-132 proteinto the receptor of one or more steroid hormones, e.g., to theprogesterone receptor or to the estrogen receptor, said receptoroptionally being complexed with the Sp1 transcription factor.

In this regard, the screening tests according to the invention arebased, in general, on selecting compounds which can increase or promotethe binding of the TReP-132 protein or of a complex comprising theTReP-132 protein to the receptor of one or more steroid hormones or elsereduce or inhibit the binding of the TReP-132 protein or of a complexcomprising the TReP-132 protein to the receptor of one or more steroidhormones.

Another object of the invention relates to methods for the selection,identification, characterization or optimization of active compounds,comprising determining the ability of a test compound to modulate theaction of the TReP-132 protein on the promoter of a target gene of theTReP-132 protein.

The screening tests according to the invention are based, in general, onselecting compounds which can modulate the expression of target genes ofthe TReP-132 protein (expression screening) or on selecting compoundswhich can modulate the binding of the TReP-132 protein to one or more ofits target genes (binding assay), that is to say, genes whose expressionis regulated by said protein.

The invention thus relates to a screening test for compounds modulatingthe formation or activity of the complex comprising a receptor of one ormore steroid hormones such as progesterone or estrogens and optionallythe TReP-132 protein and/or the Sp1 transcription factor. In thisregard, the screening tests according to the invention are based onselecting compounds which can modulate the expression of one or moretarget genes of the complex comprising a receptor of one or more steroidhormones, and optionally the TReP-132 protein and/or the Sp1transcription factor.

In the context of the invention, the target genes of the TReP-132protein are in particular the p21, p27 and p16 genes.

Another aspect of the invention relates to methods for the selection,identification, characterization or optimization of active compounds,comprising measuring the expression of a reporter gene placed under thecontrol of all or part of the promoter sequence of the TReP-132 gene, inthe presence of a test compound.

More particularly, the invention provides screening methods forselecting, identifying or characterizing biologically active compoundsor methods which can be used for validating, characterizing, optimizingor producing compounds.

The inventive methods can be implemented in different ways, in cellularor acellular tests in vitro, or in vivo.

A—Methods Based on a Binding Assay

An object of the invention is based on a method for the selection,identification, characterization or optimization of active compounds,comprising determining the ability of a test compound to modulate thebinding of the TReP-132 protein or of a complex comprising the TReP-132protein to the receptor of one or more steroid hormones. Preferably, themethod comprises determining the ability of a test compound to reduce orinhibit the binding of the TReP-132 protein or of a complex comprisingthe TReP-132 protein to the receptor of one or more steroid hormones.Even more preferably, the method comprises determining the ability of atest compound to mimic, increase or promote the binding of the TReP-132protein or of a complex comprising the TReP-132 protein to the receptorof one or more steroid hormones.

In this embodiment, one object of the invention relates to methods forthe selection, identification, characterization or optimization ofactive compounds modulating cell proliferation, comprising:

-   -   a) contacting a test compound with:        -   i) the TReP-132 protein or complex comprising the TReP-132            protein, and        -   ii) the receptor of one or more steroid hormones, and    -   b) measuring the binding of the TReP-132 protein or of the        complex comprising the TReP-132 protein to said receptor.

In the context of the invention, the steroid hormone receptor is anestrogen receptor (ER) or a progesterone receptor (PR), preferably theprogesterone receptor (PR).

Another object of the invention relates to a method for the selection,identification, characterization or optimization, in vitro or ex vivo,of active compounds reducing cell proliferation, comprising

-   a) contacting, in the presence of progesterone or estrogens, a test    compound with:-   i) respectively, the progesterone or estrogen receptor,-   ii) Sp1, and-   iii) a nucleic acid comprising all or part of the promoter of a    target gene of progesterone or estrogens, and-   b) measuring the binding of Sp1 or of the complex comprising the    progesterone or estrogen receptor and Sp1 to said promoter, or    measuring the expression of said promoter, said measurement being an    indication of the effect of the test compound on cell proliferation.

Contact is optionally carried out in the presence of the TReP-132protein or of a complex comprising the TReP-132 protein. Preferably,contact is carried out in a cell.

As indicated earlier, the steroid hormone target gene is advantageouslyselected in the group consisting of the p21, p16 and/or p27 genes.

Another object of the invention comprises a method for the selection,identification, characterization or optimization of active compoundscomprising determining the ability of a test compound to modulate thebinding of the TReP-132 protein or of a complex comprising the TReP-132protein to the promoter of target genes of the TReP-132 protein, inparticular the p27, p21 and p16 genes.

In another embodiment, the method comprises:

a) contacting the test compound in the presence of TReP-132 with anucleic acid construct comprising a sequence of all or part of a p21,p27 or p16 gene promoter or a region thereof comprising the binding siteof the TReP-132 protein or a response element of the TReP-132 protein,and

b) determining the binding of said test compound to the target nucleicacid and/or to the complex formed by the binding of TReP-132 to itsresponse element(s) or binding site(s).

In order to carry out the binding assay, it is possible to use a nucleicacid comprising all or part of the promoter sequence, and to determinein vitro the ability of a test compound to bind thereto. The sequencefragment preferably contains at least 10 consecutive nucleotides of thepromoter sequence, more preferably at least 20 consecutive nucleotidesof the promoter sequence. Moreover, it is possible to test severalfragments of the promoter sequence concomitantly.

The ability of said test compound to modulate the binding of TReP-132 tothe receptor of one or more steroid hormones or to the response elementof a target gene of the TReP-132 protein can be measured by determiningthe amount of TReP-132 bound in the presence of the test compoundcompared to said amount in the absence of the test compound or bycarrying out the reaction in the presence of labelled TReP-132 proteinand measuring the extent to which the test compound displaces thebinding of the labelled protein.

Said tests can be carried out in vitro, in any suitable device (tube,dish, flask, etc.). Optionally they can be carried out with one of thepartners being immobilized, for example the nucleic acid (column, bead,support, glass, filter, membrane, etc.).

The binding of the test compound in the context of the inventive methodscan be visualized by gel migration, electrophoresis and by other methodsbased on luminescence or using FRET (Fluorescence Resonance EnergyTransfer) or SPA (Scientillation Proximity Assay), or by any othermethod known to those skilled in the art.

B—Methods Based on Expression Screening

Another object of the invention is based on the transcriptional activityof the TReP-132 protein (transcriptional activity assay).

According to a first preferred embodiment of the invention, the methodfor the selection, identification or optimization of active compoundscomprises:

a) contacting a test compound with a reporter nucleic acid comprising areporter gene placed under the control of a promoter comprising all orpart of the TReP-132 promoter sequence, and

b) measuring the expression of the reporter gene.

Another object of the invention comprises determining the ability of atest compound to modulate the expression of one or more target genes ofthe TReP-132 protein, in particular the p21, p16 and/or p27 genes.

In this embodiment, the method comprises:

a) contacting a test compound, in the presence of the TReP-132 protein,with a reporter nucleic acid comprising a reporter gene placed under thecontrol of a promoter containing all or part of the sequence of the p21,p16 or p27 gene promoter or the binding site of the TReP-132 protein ora response element of the TReP-132 protein, and

b) determining the effect of the test compound on the expression of thereporter gene.

In a particular embodiment of the inventive methods, the possibleeffects obtained are compared with the possible effects determined by amethod carried out under the same conditions but with a nucleic acidconstruct comprising at least one inactive variant (for example a mutantcopy) of the p27, p21 or p16 promoter sequence or a region thereofcomprising the binding site of the TReP-132 protein or a TReP-132response element.

Studies have shown that TReP-132 can directly interact with DNA toregulate promoter activity. Furthermore, it has also been shown thatTReP-132 interacts with other DNA-binding proteins to regulatetranscription. Thus, TReP-132 can function as a coregulator of nuclearreceptors like SF-1 to regulate gene expression. For this reason,TReP-132 can also regulate promoter activity without interacting withDNA.

Considering that TReP-132 can function as a coregulator of nuclearreceptors, the whole protein or fragments thereof can be used in aprotein recruitment assay requiring the presence of a ligand and forscreening ligands of partner nuclear receptors. The proteincorresponding to a nuclear receptor X can be incubated with the wholeTReP-132 protein or with fragments thereof, so as to enable recruitmentof the TReP-132 polypeptide by the nuclear receptor, in the presence ofthe ligand. Depending on the type of labelling of the nuclear receptorand the TReP-132 polypeptide, different methods can be employed tomeasure protein-protein interaction or protein recruitment, includingfluorescence, scintillation counting or calorimetry. The isolatedcompounds can lead to an increase or decrease in TReP-132 recruitment inthe mechanism of expression of the gene. By using said approaches, it ispossible to screen compounds binding nuclear receptors which affect theexpression of target genes regulated by TReP-132.

A particular object of the invention relates to a method for theselection, identification, characterization or optimization of activecompounds comprising determining the ability of a test compound tomodulate the activity of the complex composed of at least the TReP-132protein and a receptor of one or more steroid hormones.

Another object of the invention relates to a method for the selection,identification, characterization or optimization of active compoundscomprising determining the ability of a test compound to modulate theexpression of target genes of the complex comprising the TReP-132protein and a receptor of one or more steroid hormones.

In this particular embodiment of the invention, the method comprises:

a) contacting the test compound with the TReP-132 protein and a reporternucleic acid comprising a reporter gene placed under the control of apromoter comprising the promoter sequence of a target gene of thecomplex comprising the TReP-132 protein and a receptor of one or moresteroid hormones, or a region of said promoter containing the bindingsite of the complex or a response element of the complex, and

b) measuring the expression of said reporter gene.

In the context of the invention, the receptor of one or more steroidhormones is an estrogen receptor (ER) or a progesterone receptor (PR).

The target genes of the TReP-132 protein are in particular the p27, p21or p16 genes.

Another object of the invention relates to a method for the selection,identification, characterization or optimization, in vitro or ex vivo,of active compounds reducing cell proliferation, comprising:

a) contacting, in the presence of progesterone or estrogens, a testcompound with:

-   -   i) respectively, the progesterone or estrogen receptor,    -   ii) Sp1, and    -   iii) a nucleic acid comprising all or part of the promoter of a        progesterone or estrogen target gene, and        b) measuring the binding of Sp1 or of the complex comprising the        progesterone or estrogen receptor and Sp1 to said promoter, or        measuring the expression of said promoter, said measurement        being an indication of the effect of the test compound on cell        proliferation.

The inventive methods can be carried out with different types of cells,promoters, reporter genes, and in different conditions, as describedhereinbelow.

Host Cell:

Some screening methods, used in the context of the invention, comprise astep of contacting the test compound with a host cell, in specificconditions allowing to determine the expression in said cell of areporter gene or of the TReP-132 protein, to possibly reveal other stepsin the synthesis of the TReP-132 protein, and thereby to obtaininformation about the effect of the test compound. Classically, theeffect of the test compound is compared with the level of reporter geneexpression or the activity of the expression product determined in theabsence of said compound.

The cells which are used can be selected from among any cells that canbe cultured in the laboratory. In a preferred embodiment of theinvention, they are mammalian cells (hepatocytes, fibroblasts,endothelial, muscle, breast cells, etc.). Even more preferably, saidcells are of human origin. They can be primary cultures or establishedcell lines. In another embodiment, it is also possible to useprokaryotic cells (bacteria), yeast cells (Saccharomyces, Kluyveromyces,etc.), plant cells, etc.

Reporter System:

A particular embodiment of the invention makes use of a reporter systemcomprising a reporter gene placed under the control of a particularpromoter. Said construct, or any cassette or vector containing it, canbe introduced into host cells, which can be used for cellular tests.

To carry out the transcriptional assay, a reporter system comprising allor part of the promoter of the target gene operationally linked to areporter gene is advantageously used.

Said reporter gene can be in particular any gene whose transcription orexpression product can be detected or measured in biological extracts.For example, it can be the gene coding for luciferase and moreparticular for firefly or Renilla luciferase, for secreted alkalinephosphatase, galactosidase, lactamase, chloramphenicol acetyltransferase (CAT), human growth hormone (hGH), β-glucuronidase (Gluc)and green fluorescent protein (GFP), etc. It shall be understood thatthe term “gene” denotes in the broad sense any nucleic acid, inparticular a cDNA, gDNA, synthetic DNA, an RNA, etc.

The reporter gene, whatever it may be, is placed under the control of apromoter containing all or part of the promoter sequence of a targetgene of the TReP-132 protein or of the complex comprising the TReP-132protein and a receptor of one or more steroid hormones, the promoter ofthe gene coding for the TReP-132 protein, the promoter of the complexcomprising the TReP-132 protein, Sp1 and a receptor of one or moresteroid hormones or a functional variant thereof, such as definedhereinabove. Preferably, it is a promoter whose differential activity inthe presence and absence of TReP-132 or a functional equivalent thereofcan be detected.

In a preferred embodiment of the invention, the reporter gene is placedunder the control of a promoter comprising the complete promotersequence of a target gene of the TReP-132 protein, a target gene of thecomplex comprising the TReP-132 protein and a receptor of one or moresteroid hormones, the promoter of the complex comprising the TReP-132protein, Sp1 and a receptor of one or more steroid hormones or thepromoter of the gene coding for the TReP-132 protein. In this regard, anobject of the invention also relates to a nucleic acid comprising areporter gene placed under the control of a promoter comprising all orpart of the p27, p21 or p16 promoter sequence, in particular the humanpromoter. The optionally selected promoter fragment advantageouslycomprises at least 10 consecutive nucleotides of said sequence,preferably at least 20, more preferably at least 30, even morepreferably at least 50. The promoter can also comprise heterologousregions, originating from other genes or promoters, such as for examplesilencer or enhancer signals, sequences conferring regulated ortissue-specific features, etc. Said particular sequences can be presentin one or more copies in the promoter (preferably 1 to 10 and morepreferably 1 to 6), upstream, downstream, or internally, in the sameorientation or in the opposite orientation. The promoter can be a hybridpromoter combining regions from other promoters, for example thepromoter of the herpes virus thymidine kinase (TK) gene, the CMV earlypromoter, the PGK promoter, the SV40 promoter, etc.

Furthermore, the different aforementioned functional domains candirectly flank each other, or be separated by nucleotides which do notsignificantly affect the functionality of the expression cassette orwhich give the system improved performance or characteristics(amplifier, silencer, intron, splicing site, etc.).

The construct can be cloned in any suitable vector, such as a plasmid,cosmid, phage, virus, etc. The construct or vector can be introducedinto a host cell by any conventional method, including in particularelectroporation, calcium phosphate precipitation, liposomes,transfectants, etc. The cells or the descendents thereof can be culturedin any suitable medium (DMEM, RPMI, etc.).

Contact:

The test compounds can be contacted with the cells at different times,according to their effect(s), their concentration, the nature of thecells and technical considerations.

Contact can be carried out on any suitable support and in particular ona plate, dish, in a tube or flask. Generally, contact is carried out ona multiwell plate which allows many different tests to be carried out atthe same time. Typical supports include microtitration plates and moreparticularly plates with 96 or 384 wells (or more), which are easy tomanipulate.

Depending on the support and the nature of the test compound, variableamounts of cells can be used to carry out the aforementioned methods.Classically, between 10³ and 10⁶ cells are contacted with a particulartest compound, in a suitable culture medium, and preferably between 10⁴and 10⁵ cells. As an example, in a 96-well plate, 10⁵ cells can beincubated in each well with a desired quantity of a test compound; in a384-well plate, fewer than 10⁵ cells and typically between 1×10⁴ and4×10⁴ cells are generally incubated in each well with the test compound.

The quantity (or the concentration) of test compound can be adjusted bythe user according to the characteristics of said compound (itstoxicity, ability to penetrate cells, etc.), the number of cells, thelength of the incubation period, etc. Generally, the cells are exposedto concentrations of test compounds ranging from 1 nM to 1 mM. Of courseit is possible to test other concentrations without deviating from theinvention.

Also, each compound can be tested in parallel at differentconcentrations.

Different adjuvants and/or vectors and/or products facilitating thepenetration of the compounds into the cells such as liposomes, cationiclipids or polymers can also be used, when necessary.

Contact is typically maintained for several minutes to several hours,generally between 1 and 48 hours. In particular, when the assaycomprises the expression of a reporter gene, the cells and variousreagents should preferably remain in contact long enough to allow denovo synthesis of the reporter gene expression product.

Measurement of Effect:

The measurement or demonstration of an effect of the test compound canbe carried out in different ways, according to the assay used.

Methods for detection of binding in vitro are mentioned hereinabove.

Transcriptional activity assays comprise a step of determining anddetecting the expression of the reporter gene.

This may be a determination of transcriptional activity. To this end,total RNA is extracted from cultured cells in the experimentalconditions on the one hand and in a control situation on the other hand.Said RNA is assayed (or used as probe) to analyze changes in expressionof the reporter gene(s).

It can also be a matter of visualizing or assaying the reporter geneexpression product. Said visualization (or said assay) can be achievedby different methods the nature of which depends on the type of reportergene used. For example, the measurement can correspond to an opticaldensity or a fluorescence emission in the case where the β-galactosidaseor luciferase gene is used as reporter gene.

The expression of the reporter gene can also be measured in terms of thehydrolysis of a substrate of the reporter gene expression product, suchas for example a substrate of β-lactamase. One can mention in particuarany product containing a β-lactam nucleus and the hydrolysis of whichcan be measured. Preferred substrates are those specific of β-lactamase(i.e., they are generally not hydrolyzed in mammalian cells in theabsence of β-lactamase), those which are nontoxic to mammalian cellsand/or whose hydrolysis product can be easily measured, for example bymethods based on fluorescence, radioactivity, an enzymatic activity orany other method of detection.

Even more preferred substrates are the radiometric substrates. Thehydrolysis of said substrates can be directly correlated with theactivity of the reporter gene product by the number of cells.

The expression product can also be measured by immunological orimmunoenzymatic methods, involving a specific antibody for example. Saidsystem is particularly suited to assaying for example the TReP-132protein synthesized by a cell treated or not treated with a testcompound.

In a general manner, the presence of the reporter gene product (or thehydrolysis product of the substrate) can be determined by conventionalmethods known to those skilled in the art [fluorescence, radioactivity,OD, luminescence, FRET (see WO 0037077), SPA, biochips, immunologicalmethods, etc.]. Generally, one determines the activity of a testcompound in a cell and said effect is compared with the level ofactivity in the absence of test compound or with a mean value determinedin the absence of any test compound.

Hydrolysis is measured essentially by measuring (or determining therelative amount) of the hydrolysis product contained in each reactionsample with the aid of different methods known to those skilled in theart, including detection of fluorescence, radioactivity, color, anenzymatic activity, an antibody-antigen immune complex.

A secondary test can be carried out whereby the selection of thecompounds can be validated, for example by determining cellproliferation in culture or in animals, or by comparison with untreatedcells or animals.

The invention is particularly adapted to the selection, identificationor characterization of a large number of compounds. Said simple andefficient screening can be accomplished in a very short time. Inparticular, the described methods can be partially automated, therebyenabling the efficient and simultaneous screening of many differentcompounds, either in the form of a mixture or separately.

The aforementioned methods for the selection, identification andcharacterization of test compounds according to the invention can beused for the selection, identification and characterization of compoundswhich can inhibit cell proliferation and/or regulate the cell cycle.

Other objects of the invention relate to a method for detecting apredisposition, a method of diagnosis, a method of monitoring andmeasuring the aggressiveness of a proliferative disease comprisingmeasuring the binding of the TReP-132 protein to one or more steroidhormones in a sample taken from a mammal, in particular a human. Asindicated earlier, the receptor of one or more steroid hormones ispreferably a progesterone receptor (PR) or an estrogen receptor (ER).

The aforementioned methods can comprise or involve solely the assay ofthe TReP-132 protein.

A particular object of the invention further relates to a method fordetecting a predisposition, a method of diagnosis, monitoring andmeasuring the aggressiveness of a proliferative disease comprisingdetecting, in a patient, the overexpression of estradiol, the reductionof progesterone or a decrease in the expression of the CDKIs p21, p16and/or p27.

Test Compounds

The compounds which can be identified with the aid of a method accordingto the invention can be compounds of different nature, structure andorigin, in particular biological compounds, nuclear factors, cofactors,chemical, synthetic compounds, etc. The invention can therefore be usedwith any type of test compound. For instance, the test compound can beany product which is isolated or in a mixture with other products. Thecompound can be defined in terms of structure and/or composition or bedefined in terms of function. For example, the compound can be anisolated and structurally defined product, an isolated product withundefined structure, a mixture of known and characterized products or anundefined composition comprising one or more products. Hence one or morecompounds can be tested, in a mixture or separately. For example, saidundefined compositions can be samples of tissues, biological fluids,cell supernatants, plant preparations, and the like. The test compoundscan be selected from among an inorganic or organic product and inparticular a polypeptide; protein or peptide, a nucleic acid, lipid,polysaccharide and a chemical or biological compound. It can be forexample a nuclear factor, a cofactor or any mixture or derivativethereof. The compound can be natural or synthetic and can include acombinatorial library, a clone or a library of nucleic acid clonesexpressing one or more DNA-binding polypeptides, etc.

Pharmaceutical Composition

A particular object of the invention relates to a pharmaceuticalcomposition comprising a compound increasing or mimicking the activityof the TReP-132 protein and a pharmaceutically acceptable excipient.

Preferably, said pharmaceutical composition is used in combination witha hormone for a simultaneous, separate or sequential administration.

Even more preferably, the pharmaceutical composition according to theinvention comprises the TReP-132 protein or an analog thereof in apharmaceutically acceptable excipient.

The invention relates to a pharmaceutical composition comprising acompound modulating, in particular mimicking, increasing or promoting,the binding of the TReP-132 protein or of a complex comprising theTReP-132 protein to the receptor of one or more steroid hormones and apharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition comprises a compoundstimulating the binding of the TReP-132 protein to the receptor of oneor more steroid hormones and a pharmaceutically acceptable excipient.Even more preferably, the pharmaceutical composition comprises acompound increasing the binding of the TReP-132 protein to theprogesterone receptor (PR) or to the estrogen receptor (ER).

Another object of the invention relates to a pharmaceutical compositioncharacterized in that it comprises, in a pharmaceutically acceptablevehicle, a compound stimulating the activity of the complex comprisingthe TReP-132 protein and a receptor of one or more steroid hormones.

Another particular object of the invention relates to a pharmaceuticalcomposition, characterized in that it comprises, in a pharmaceuticallyacceptable excipient, a compound modulating, in particular mimicking,increasing or promoting, the action of the TReP-132 protein on one ofits target gene promoters, in particular that of the p21, p27 or p16gene.

The invention also relates to a pharmaceutical composition comprising,in a pharmaceutically acceptable vehicle, a nucleic acid coding for theTReP-132 protein. Preferably, the nucleic acid is cloned in anexpression vector, preferably a plasmid or viral vector.

Another object of the invention relates to a pharmaceutical compositioncomprising, in a pharmaceutically acceptable excipient, a compoundmodulating the formation or activity of the complex comprising theTReP-132 protein, Sp1 and a receptor of one or more steroid hormones.

A particular object of the invention relates to a pharmaceuticalcomposition comprising, in a pharmaceutically acceptable excipient, acompound selected in the group consisting of the TReP-132 protein, ananalog thereof, a compound increasing or mimicking the activity ofTReP-132 and a compound identified with the aid of the one of themethods according to the invention, combined with one or more hormones,in view of a simultaneous, separate or sequential administration for thetreatment of a proliferative disease.

In a preferred manner, the pharmaceutical composition according to theinvention comprises a compound identified with the aid of a method suchas described hereinabove. Even more preferably, the selected compound isTReP-132.

In a preferred manner, the hormone combined with the selected compoundis progesterone.

The pharmaceutical compositions according to the invention, intended forthe treatment of proliferative diseases, can be prepared in particularaccording to a method comprising a step involving a method such asdescribed earlier.

The pharmaceutical compositions according to the inventionadvantageously comprise one or more pharmaceutically acceptableexcipients or vehicles. Examples include saline, physiological,isotonic, buffered solutions and the like, compatible withpharmaceutical use and known to those skilled in the art. Thecompositions can contain one or more agents or vehicles selected in thegroup consisting of dispersants, solubilizers, stabilizers, surfactants,preservatives, and the like. Also, the inventive compositions cancomprise other active ingredients or agents.

The compounds or compositions of the invention can be administered indifferent ways and in different forms. The inventive compounds aretypically administered by the systemic route, preferably locally.Advantageously, intratumoral injection as well as injection in a regionclose to the tumor or irrigating a tumor can be mentioned.

Different doses can be employed, according to the compound, the numberof administrations, combination with other active ingredients, the stageof disease, etc. The invention discloses an advantageous and efficientnovel strategy for treating proliferative diseases. In fact, theinvention is based on the characterization of a natural metabolicpathway which can be subjected to selective interventions.

Other advantages and applications of the invention will become apparentin the following examples, which are given for purposes of illustrationand not by way of limitation.

LEGENDS TO THE FIGURES

FIG. 1: Effect of the TReP-132 protein on formation of G418-resistantcolonies.

FIG. 2: Expression of the TReP-132 protein in MCF-7 breast cancer cells,estrogen-dependent.

FIG. 3: Induction of TReP-132 by progesterone and suppression ofprogesterone inhibition of T47-D cell proliferation following TReP-132knockdown by using small interfering RNA (“siRNA”). (T47-D cells weretreated for the indicated times with 30 nM progesterone). Experimentswere carried out in triplicate, and values represent the mean ±SD of arepresentative experiment.

FIG. 3A: Analysis of TReP-132 mRNA levels by quantitative RT-PCR.

FIG. 3B: Cells transfected with control siRNA or TReP-132 siRNA (400 ng)at 0 and 3 days were recovered at days 2, 4 or 6 and counted. (ns: notsignificant −*, p<0.05; **, p<0.0; ***, p<0.001).

FIG. 4: Bidirectional Tet-Off expression system.

FIG. 5: Induction of TReP-132 protein expression in the absence ofdoxycycline.

FIG. 6: Inhibition of cell proliferation by the TReP-132 protein.

FIG. 7: Effet of TReP-132 protein induction on the cell cycle G₁→Stransition.

FIG. 8A, 8B: Expression of TReP-132 mRNA after cell synchronization atthe end of G₁ phase.

FIG. 9: Effect of TReP-132 on G₁ CDK activities and pRb phosphorylationlevels.

FIG. 9A: Kinase activities of each immunoprecipitated cyclin or CDK werequantified using pRb as substrate to measure cyclin D1/CDK6 activity, orhistone H1 to measure cyclin A/CDK2 activity. The levels ofphosphorylation of histone H1 or pRb in induced HTO cells (HeLa Tet Off)are expressed in relation to the levels in non-induced HTO cells(corresponding to level 1.0).

FIG. 9B: Protein extracts (20 μg) of HTO cells expressing GFP or GFP andTReP-132 under the control of doxycycline, incubated or not with dox forthe indicated times, were analyzed by western blot using anti-pRbantibodies directed against all pRb forms (hyperphopshorylated,phosphorylated and hypophosphorylated) or only against pRbphosphorylated at Ser 780, a target residue of cyclin D1/CDK4.

FIG. 10: Effect of induction of TReP-132 protein expression on thetranscription of key cell cycle regulatory genes (CDK and CDKI).

FIG. 11: TReP-132 protein activates the p16, p21 and p27 promoters.

FIG. 12: Synergistic action of TReP-132 protein with Sp1 and p300 on p21promoter activation.

FIG. 13: Induction of p21 and p27 promoter activity by TReP-132involving an interaction with Sp1 in HeLa cells.

FIGS. 13A and 13B: Luciferase reporter constructs under the control ofthe deleted or mutant p21 and p27 promoters were cotransfected in HeLacells with Sp1 (0.1 μg) and/or TReP-132 (0.3 μg) expression vectors.Cell lysates were then assayed for luciferase activity. The resultsrepresent the levels of luciferase activity, the activity of the−2320p21 Luc and −3568p27 Luc reporter constructs being arbitrarily setto 1.0. Arrows indicate transcription initiation sites.

FIG. 13C: HTO-GFP or HTO-GFP/TReP-132 cells incubated or not with Doxfor 48 hours were recovered for extraction of soluble chromatin. Theimmunoprecipitations (IPs) were carried out with anti-Flag antibody.Control PCRs were carried out without DNA (H₂O) or with unprecipitatedgenomic DNA (input). The DNA extracts were amplified using the followingprimers: region −117/−65 of the p21 gene promoter, region distal to thep21 gene promoter (1 kb upstream of promoter), region −549/−511 of thep27 gene promoter, region distal to the p27 gene promoter (1 kb upstreamof promoter) or a region of the beta-actin gene.

FIG. 13D: GST-TReP-132 proteins were incubated with ³⁵-[S]-Sp1 andimmobilized on a glutathione-Sepharose column. Specific interactionswere analyzed by comparison with the results obtained by incubating GSTalone with ³⁵-[S]-Sp1. Sp1 input: 1/10^(th) of radiolabelled Sp1 levelused in the incubations.

FIG. 14: Increase in progesterone-dependent activation of the p21 andp27 gene promoters by TReP-132, via increased binding to Sp1 bindingsites in T47-D cells.

FIGS. 14A and 14B: T47-D cells were cotransfected with reporterconstructs containing the p21 (FIG. 14A) or p27 (FIG. 14B) promoter.Twelve hours post-transfection, the cells were incubated with 30 nMprogesterone or ethanol for 34 hours. The cell lysates were then assayedfor luciferase activity. Luciferase activity of the −93p21 Luc and−549p27 Luc constructs represent level 1. Arrows indicate transcriptioninitiation sites.

FIG. 14C: Chromatin was from T47-D cells treated or not with 30 mMprogesterone. IPs were carried out with anti-TReP-132 antibodies(columns 2-3). Controls were DNA-free PCR products (column 1, H₂O), PCRproducts of unprecipitated genomic DNA (columns 2 and 3, input),immunoprecipitations with no antibody (columns 4 and 5, no Ab) or withirrelevant antibody (columns 6 and 7, anti-HA). The extracted DNA wasamplified using the same primers as in FIG. 13C.

FIG. 14D left: GST-TReP-132 immobilized on glutathione-Sepharose wasincubated with ³⁵-[S]-methionine-labelled PR, in the absence or withincreasing concentrations of progesterone (0-10 μM). Specificinteractions were analyzed by comparison with the results obtained byincubating GST with ³⁵-[S]-PR, in the presence of 100 μM progesterone(GST).

FIG. 14D right: GST-TReP-132 wt, GST-TReP-132 ml (mutated at the LRQLLsite) and GST-TReP-132 m2 (mutated at the LEMLL site) were analyzed forinteraction with ³⁵-[S]-PR as described earlier, in the presence of 10μM progesterone. Input PR: 1/10^(th) of the radiolabelled PR level usedin the experiments.

FIG. 15: Analysis of the interaction between TReP-132 and the estrogenreceptor (ER).

GST-TReP-132 wt was analyzed in terms of the interaction with the³⁵[S]-ER, as described previously.

EXAMPLES Materials and Methods

1. Plasmids.

The TReP-132-HA vector, containing the entire TReP-132 cDNA tagged withthe HA epitope at the 3′ end, was constructed by PCR amplification ofTReP-132 cDNA with oligonucleotides introducing a KpnI restriction siteat the 5′ end and a HA epitope and XbaI site at the 3′ end. The PCRproducts were digested with KpnI and XbaI and subcloned into the pcDNA3expression vector (Invitrogen, Carlsbad, Calif.). The GST-TReP-132vector was generated by in-frame fusion of the TReP-132 coding regiondownstream of the GST sequence in the pGEX-2TK vector (AmershamPharmacia Biotech, Baie d'Urfé, Quebec, Canada). The mutantsGST-TReP-132 ml and GST-TReP-132 m2 were constructed using theQuickChange site-directed mutagenesis kit (Stratagene), by mutation ofleucines to alanines in NR-boxes (Nuclear Receptor box) 1 and 2 (Gizardet al., 2002b). The pBI-EGFP plasmid which contains atetracycline-responsive bidirectional promoter enables coexpression of agene of interest with green fluorescent protein (EGFP for “EnhancedGreen Fluorescent Protein”). This plasmid, and the pBI-EGFP-Luc controlplasmid expressing both GFP and luciferase, were obtained from ClontechLaboratories, Palo Alto, Calif. The pBI-EGFP-TReP-132 plasmid was formedby directly cloning the entire TReP-132 cDNA obtained by PCR into theNheI site of pBI-EGFP. All constructs were verified by dideoxynucleotidesequencing. The pcDNA3-Sp1 expression vector (Blais et al., 2002) andthe human gene p21^(WAF1) reporter plasmid containing the −1560/+34fragment (p21-Luc −1560/+34) subcloned into the pGL3 vector weregenerously provided by Dr. Monté (IBL, France). The −2320p21 Luc and−154p21 Luc constructs were generously provided by Drs Kraft and Biggs(Division of Oncology, University of Colorado, Health Sciences Center,Denver), the −93p21 construct Luc and mutants thereof by Dr. Wang (Dept.of Pharmacology, Duke University Medical Center, NC), and the humanp27^(KiP1) reporter construct subcloned into the pGL2 basic vector(Promega) by Dr. Toshiyuki Sakai (Kyoto Prefectural University ofMedecine, Japan). The 5′ region of the human p16^(CDKN2) gene containingthe nucleotide sequence from −1 to −869 relative to the ATG, subclonedbetween the KpnI and BgIII sites of pGL2 was kindly provided by Dr.Gordon Peters. The series of p16^(CDKN2) reporter vectors wereconstructed by this team by subcloning different PCR fragments generatedwith 5′-specific oligonucleotides of the gene.

2. Cell Cultures.

Human cervical HeLa cells and breast tumor cell lines T47-D and MCF-7were obtained from the ATCC (American Type Culture Collection, Manassas,Va.) and were cultured in monolayers. HeLa cells were grown in DMEMmedium (Dulbecco's modified Eagle's medium) (Life Technologies,Gaithersburg, Md.), MCF-7 cells in DMEM-F12 medium (composed of an equalmixture of DMEM and Ham's F-12 medium), and T47-D cells in RPMI 1640.All media were supplemented with 10% fetal calf serum (Hyclone, Logan,Utah), 2 mM glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin(Life Technologies, Ontario, CA). For MCF-7 cells, the medium wassupplemented with 10⁻⁹ M estradiol (E2) (Sigma-Aldrich CA Ltd.).

For transfection assays, HeLa and T47-D cells were incubated for 24hours in 24-well dishes at an initial density of 1.5×10⁴ and 1.5×10⁵cells per well, respectively. HeLa cells were then transfected withExGen 500 (MBI Fermentas, Flamborough, Ontario, Canada) at a ratio of 4μl of ExGen 500 per 0.5 μg of DNA. T47-D cells were transfected withFuGENE 6 (Roche Molecular Biochemicals, Laval, Canada) at a ratio of 3:1(FuGENE:DNA).

The pGL2 and pGL3 reporter constructs (100 ng) were cotransfected withthe indicated amounts of expression vectors and 10 ng of the controlplasmid pRL.Null. The amount of plasmid was then normalized with theempty vector pcDNA3. After 12 hours of transfection, the medium waschanged and cells were incubated for an additional 10 and 34 hours,respectively. During this period, T47-D cells were cultured in thepresence and absence of progesterone (30 nM). The cells were thenharvested and cell lysates (20 μl) were assayed for luciferase activity(Dual Luciférase™ Reporter Assay System, Promega, Madison, Wis., USA) ina Berthold LUMAT LB9501 luminometer. The transfections were performed intriplicate and each experiment was repeated at least three times.

3. Colony Formation Assays.

HeLa cells were seeded in 12-well plates at an initial density of100,000 cells per well, incubated for 24 hours, then transfected witheach of the following plasmids: pcDNA3, pcDNA3-Luc, pcDNA3-SF-1, orpcDNA3-TReP-132, each containing the neomycin resistance gene. Thetransfections were carried out in triplicate with ExGen 500 (MBIFermentas, Flamborough, Ontario, Canada) at a ratio of 5 μl of ExGen 500per 1 μg of plasmid. After 12 hours of transfection, the medium waschanged and the cells were incubated for an additional 8 hours. Thecells were then trypsinized and transferred to a 100 mm petri dish.After 48 hours, the medium was supplemented with 400 μg/ml of theselection agent G418 (Sigma-Aldrich CA Ltd.). Distinct colonies could bedetected after two weeks of selection with G418. The cells were thenrinsed with PBS and fixed with a mixture of 10% acetic acid/10% methanolfor 15 min, then colored with crystal violet (Sigma-Aldrich CA Ltd.).

4. RNase protection assays in MCF-7 cells.

The radiolabelled TReP-132 probe was prepared as follows: thepcDNA3-TReP-132-HA plasmid was first linearized by digestion with therestriction enzyme Narl (New England Biolabs, Beverly, Mass.) beforepriming in vitro transcription from the T7 promoter in the presence of[α-³²P]UTP ribonucleotide in the conditions specified in the MAXIscriptkit (Ambion, Austin, Tex.). This generated a 226 bp cRNA probecontaining 174 nucleotide bases located at the 3′ end of TReP-132 and 52bases originating from the polylinker and including the HA tag. The 18Sriboprobe used for normalization was generated from the human pTRI-18Svector (Ambion, Austin, Tex.) and protects an 80 bp fragment.Ribonuclease protection assays were carried out with the Ribonuclease IIProtection kit (Ambion, Austin, Tex.) according to the manufacturer'sprotocol. Thirty micrograms of RNA from MCF-7 cells were hybridized withthe two probes for 16 hours at 45° C. and the hybrids were then digestedfor 45 min at 37° C. in RNase A/RNase T1 solution diluted 1:75 indigestion buffer. The samples were separated on a polyacrylamide-6% ureagel.

5. Reverse transcription and quantitative PCR.

Total RNA from T47-D cells treated or not with progesterone was reversetranscribed and TReP-132 transcript levels were measured by quantitativeRT-PCR as described in Gizard et al. (2004) using primers specific forTReP-132 (5′-gtcaacaatatggcccaggtg-3′ and 5′-gccagaggctgctggtcgtc-3′).

6. Silencing of TReP-132 Expression by the Small Interfering RNA (siRNA)Method.

The sense 5′-aacatgtttgagttgccaggcctgtctc-3′ and the antisense5′-aacctggccaactcaaacatgcctgtctc-3′ oligonucleotides were synthesized(Eurogentec) and used to generate double-stranded siRNA specific forTReP-132 (21 bp corresponding to amino acids 861-881 upstream of theinitiation codon of the human TReP-132 gene). The nonsilencing siRNAoligonucleotide from Qiagen, which does not target any mammalian gene,was used as negative control. Transfection was carried out by using theGeneSilencer reagent (Gene Therapy Systems, San Diego, USA) as directedby the manufacturer. Transfection efficiency was verified by FACSanalysis (Fluorescence-Activated Cell Sorting) (see below). QuantitativePCR showed a decrease of 75±12% in TReP-132 mRNA levels. Statisticalanalyses were carried out with the nonparametric Kruskal-Wallis test.

7. Synchronization of HeLa Cells in G₁ phase.

HeLa cells were synchronized in G₁ phase at the G₁→S transition by adouble thymidine block as described by Cao et al. (1991) and Tobey etal. (1967) with some modifications. Addition of an excess of thymidineinhibits the enzymatic reaction catalyzing the formation ofdeoxycytidine triphosphate from cytidine-5′-phosphate and prevents DNAreplication, which occurs in the S phase. HeLa cells were first seededat 10⁶ cells per petri dish (100 mm diameter) and cultured for 24 hours.Thymidine was added at 5 mM final concentration and the cells wereincubated for 14 hours. This time interval was calculated to be slightlylonger than the sum of the G₂, M and G₁ phases for HeLa cells. The cellswere then synchronized to enter S phase by removing thymidine from themedium for 9.5 hours, which is longer than the duration of the S phase.The cell cycle was halted prior to entering S phase by again addingthymidine to the medium for 5 hours. The time at which thymidine wasremoved corresponds to time point zero in these experiments.

8. Inducible TReP-132 Expression by the Tet-Off System.

HeLa Tet-off cells expressing the tTA transactivator under the controlof tetracycline or doxycycline (Dox) (Clontech Laboratories, Palo Alto,Calif.) were stably transfected with the pBI-EGFP-TReP-132 vector or thepBI-EGFP control vector. In order to select stably transfected cells,the pcDNA6 plasmid (Invitrogen, Ontario, CA) containing the blasticidinresistance gene was cotransfected at a ratio of 1:50. For thesetransfections, the cells were first seeded in 6-well plates at aninitial density of 1×10⁵ cells per well, incubated for 24 hours, thentransfected with ExGen 500 (MBI Fermentas, Flamborough, Ontario, CA) ata ratio of 5 μl of ExGen 500 for a total of 1 μg of plasmid such asdescribed hereinabove in the section “Colony formation assays”. 48 hourspost-transfection, cells were selected for 2 weeks with blasticidin(Sigma-Aldrich CA Ltd.). Clearly visible colonies were then were pickedand subcloned on 12-well plates to enable growth. It should be notedthat the cells were continuously kept in the presence of doxycycline soas to repress the transcription of genes under control of the tTAtransactivator.

9. Cell Sorting by FACS (Fluorescence-Activated Cell Sorting).

To determine the efficiency of the doxycycline-controlled inducibleexpression system in clones stably transfected with the pBI-EGFP-TRePvector (system described hereinabove), the fluorescence of each cellwithin a given population was measured individually by flow cytometry onan EPICS XL (Beckman Coulter, Mississauga, CA). The following parameterswere measured: FS (Forward scatter), SS (side scatter) and fluorescenceemission at 520 nm after excitation with an argon laser at 488 nm. Cellsexpressing GFP were then sorted on an EPICS ELITE ESP (Beckman Coulter,Mississauga, CA).

10. Measurement of Cell Proliferation.

The cell proliferation rate was determined by the cellular DNA contentusing the fluorochorome DABA (dimethylaminobenzaldehyde). The medium wasremoved and cells were dehydrated by addition of 150 μl of methanol toeach well. After complete evaporation, cells were incubated for 1 hourat 60° C. with 150 μl of DABA solution prepared as follows: 2 g of DABAwere dissolved in 10 ml of 1 N hydrochloric acid (HCl), followed byaddition of Norit A Charcoal at 1 g/10 ml. The mixture was shaken,centrifuged for 30 minutes at room temperature, then filtered on a 0.45micron filter. After incubation, the plates were placed on ice for 10min and 1.25 ml of 1 N HCl was added to each tube. Fluorescence wasmeasured on a Perkin-Elmer LS-2B Filter Fluorimeter at an excitationwavelength of 400 nm and an emission wavelength of 508 nm. The DNAcontent was calculated from a standard curve of known quantities ofsalmon sperm DNA.

11. Cell Cycle Analysis by Flow Cytometry.

To evaluate the percentage of cells in the G₀/G₁, S, or G₂/M phase, flowcytometry profiles were determined on propidium iodide stained DNA fromsaid cells. To this end, the cells were trypsinized, centrifuged,resuspended in 300 μl of PBS and cooled on ice. 700 μl of 95% ethanolwere then added dropwise while stirring the cells gently on a vortex,and the cells were fixed in ice for 30 min. After centrifugation andwashing with PBS, the cells were incubated in 500 μl of PBS containing40 U/ml of RNase (“DNAse free”, Roche Diagnostics, Laval, Quebec, CA)and 50 μg/ml of propidium iodide (Sigma-Aldrich CA Ltd.) for 30 min atroom temperature in the dark. DNA measurements were carried out on anEpics XL (Beckman Coulter, Mississauga, CA) at an excitation wavelengthof 488 nm and an emission wavelength of 620 nm. The percentage of cellsin the G1, S, and G₂/M phase was quantified with Multicycle AV software(Phoenix Flow System, San Diego, Calif.) (28).

12. Transfections of HeLa and T47-D Cells.

TReP-132 activation of the p21, p27 and p16 cyclin kinase inhibitorpromoters was studied by transfection of HeLa and T47-D cells. HeLacells were seeded in 24-well plates at an initial density of 15,000cells per well and transfected the next day with ExGen 500 (MBIFermentas, Flamborough, Ontario, Canada) at a ratio of 4 μl of ExGen 500per 0.5 μg DNA. T47-D cells were seeded at a density of 100,000 cellsper well and transfected the next day with FuGENE 6 reagent (RocheMolecular Biochemicals, Laval, Canada), at a ratio of 3 μl of FuGENE per1 μg of DNA. After a 12-hour incubation at 37° C., the medium wasreplaced and the cells were incubated for another 8 hours, then lysedfor 15 minutes at room temperature in a solution containing 0.8% TritonX-100, 25 mM glycylglycine pH 7.8, 15 mM MgSO4, and 4 mM EGTA.Luciferase activity was assayed on a Berthold Lumat LB9501 luminometer(Berthold Detection Systems GmbH, Pforzheim, Germany) using theDual-Luciferase™ reporter assay system kit (Promega Corporation,Madison, Wis.) enabling the simultaneous assay of firefly and Renillaluciferase activity (encoded by plasmid pRL-null for normalization). Forall transfections, 0.1 μg of reporter plasmid containing the promoterunder study (p21^(WAF1/CIP1), p27^(Kip1), or p16^(CDKN2)) and 0.01 μg ofpRL-null were used. The amounts of TReP-132, p300, and Sp1 expressionvectors varied according to the experiment and are indicated in the“Results” section. Each experiment included a control transfection withpcDNA3-EGFP and flow cytometry analysis confirmed that at least 60% ofcells were transfected.

13. RNase Protection Assays in HeLa Cells.

The radiolabelled TReP-132 probe was prepared as described earlier: thepcDNA3-TReP-132-HA vector was first linearized by digestion with therestriction enzyme Narl (New England Biolabs, Beverly, Mass.) prior topriming in vitro transcription from the T7 promoter in the presence of[α-³²P]UTP-radiolabelled ribonucleotide in the conditions specified inthe MAXIscript kit (Ambion, Austin, Tex.). A 226 bp cRNA probe wasgenerated, containing 174 nucleotide bases located at the 3′ end ofTReP-132 and 52 bases originating from the polylinker and including theHA tag. The 18S riboprobe used for normalization was generated from thehuman pTRI-18S vector (Ambion, Austin, Tex.) and protected an 80 bpfragment. The ribonuclease protection experiments were carried out withthe Ribonuclease II Protection kit (Ambion, Austin, Tex.) as directed bythe manufacturer. Thirty micrograms of RNA from MCF-7 cells werehybridized with the two probes for 16 hours at 45° C. and the hybridswere then digested for 45 min at 37° C. in RNase A/RNase T1 solutiondiluted 1:75 in digestion buffer. The samples were separated on apolyacrylamide-6% urea gel.

All radiolabelled probes specific for the p130, Rb, p107, p53, p57, p27,p21, p19, p18, p14/p15, L32, and GAPDH genes were labelled with[α-³²P]UTP-ribonucleotide (PerkinElmer Life Sciences, Woodbridge,Ontario, CA) in the conditions specified in the “hCC-2 Muti-ProbeTemplate Set” kit (Pharmingen, Mississauga, Ontario, CA). Ribonucleaseprotection experiments were carried out with the Ribonuclease IIIProtection kit (Ambion, Austin, Tex.). Ten micrograms of HeLa cell RNAwere hybridized with the radiolabelled probe mixture at 56° C. and thendigested for 45 min at 30° C. in RNase A/RNase T1 solution diluted1:1000 in digestion buffer. The samples were separated on apolyacrylamide-6% urea gel.

14. Preparation of Protein Extracts, Analysis by Western Blot andCo-Immunoprecipitation.

Cells were washed twice with cold PBS (Phosphate Buffered Saline), thenharvested either in cold RIPA buffer (1×PBS, 1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitors) forco-immunoprecipitation analysis, or in lysis buffer (50 mM Tris, pH 8.0,0.1% IGEPAL (Sigma)) for kinase assays. The lysates were incubated for30 min on ice and cell debris was eliminated by centrifugation. Theextracts were then aliquoted and stored at −80° C. For western blotanalysis, RIPA or lysate buffer was used. For immunoprecipitations,whole cell lysates (1 mg) in RIPA buffer were preincubated with 0.1 μgof mouse or rabbit IgG and 20 μl of protein A-Sepharose beads (AmershamBiosciences, Uppsala, Sweden) for 30 min at 4° C. The lysates (1 mg)were then centrifuged and immunoprecipitated for 1 hour at 4° C. withanti-CDK2 (sc-163) (Santa Cruz), anti-CDK6 (RB-017), anti-cyclin A(RB-010) or anti-cyclin D1 (MS-395) (Microm France, Francheville)antibody, then incubated overnight at 4° C. with protein A-Sepharose.The immunoprecipitated proteins were washed four times with lysisbuffer, then resuspended in electrophoresis buffer. Protein samples(20-100 μg) were heated at 95° C. for 5 min, then separated by SDS-PAGEand transferred to nitrocellulose membranes previously saturated withpowdered milk diluted in TBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl)containing 0.2% Tween 20 (Sigma) and then incubated (2-4 hours at roomtemperature or overnight at 4° C.) with the antibodies anti-p21 (OP64)(Merck Eurolab, Fontenay-sous-Bois, France); anti-p27 (K25020,Transduction Laboratories, Lexington, Ky.); anti-pRb (554136,recognizing all phosphorylation states of pRb); anti-ppRb-Ser780(sc-12901) specific for a pRb phosphorylated on Ser 780; anti-PR(MS-298-P) and anti-beta-actin (sc-7210) as control. The membranes werewashed, then incubated with mouse or rabbit HRP-coupled secondaryantibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) for 45min. The blots were then visualized with ECL reagent and quantified withSCANWISE and Perfect-IMAGE V-5.3 software (CLARA VISION, France).

15. Kinase Assays.

The kinase activities on histone H1 or Rb protein in theimmunoprecipitates were measured as follows: the immunoprecipitates werefirst washed once with 1 ml of assay buffer (50 mM Tris-HCl, pH 7.4, 10mM MgCl₂, 1 mM dithiothreitol (DTT)), then resuspended in 20 μl of assaybuffer containing 1 mg/ml casein (Sigma) and 1 μg of histone H1 or Rbprotein (769) (sc-4112) as substrate. After a 5-min preincubation at 30°C., the reaction was initiated by adding 1 μM ATP and 5 μCurie of[gamma-³²P] ATP, then continued at 30° C. for 1 hour.

The reaction was stopped by addition of 15 μl of electrophoresis buffer(3×). The samples were run on a 10% SDS-PAGE gel. The gel was dried andautoradiographed. Phosphorylated Rb and histones were quantified bydensitometry.

16. In Vitro Binding Assays.

In order to evaluate the interaction of TReP-132, Sp1, ER and PR, the wtand mutant GST-TReP-132 fusion proteins were expressed, then immobilizedon glutathione-Sepharose as described (Frangioni and Neel, 1993; Monteet al., 1998). The ³⁵S-[Sp1], ³⁵S-[ER] and ³⁵S-[PR-B] proteins weresynthesized in vitro using a rabbit reticulocyte lysate and T7 RNApolymerase system as directed by the manufacturer (Promega Corporation,Madison, Wis.). Proteins which bound, then detached from the Sepharoseafter being heated to boiling in SDS buffer were then separated on a 10%SDS-PAGE gel. The gels were stained with Coomassie blue, dried andautoradiographed.

17. Chromatin Immunoprecipitation (ChIP).

HTO cells incubated in the presence of Dox or T47-D cells were grown to70% confluence in petri dishes (150 mm diameter). For HTO cells,doxycycline was removed from half of the dishes 48 hours before lysis.T47-D cells were incubated with 30 nM progesterone or with ethanolcontaining 0.2% BSA-RPMI for 2.5 hours before lysis. Cell lysates werethen sonicated on ice, 15 times for 15 seconds at 45 second intervals. Avolume of lysate corresponding to 4×10⁶ cells was immunoprecipitated byusing 4 μg of antibody (indicated earlier) and an anti-HA antibody asnegative control. The same volume was set aside for later purificationof genomic DNA. One-twentieth of the DNA extract was then amplified byPCR for 35 cycles (30 sec at 92° C., 30 sec at 55° C. and 30 sec at 72°C.) using the primers: for p21, 5′-ggcactcttgttcccccaggc-3′ andaccatccccttcctcacctg-3′ (Sp1 response element on p21) or5′-gcacactgacgcagcacacag-3′ and 5′-cagtttgagaagcagccacct-3′ (distal p21element); for p27, 5′-aggccagccagagcaggtttgt-3′ and5′-ggaggagatccattggttgcgg-3′ (Sp1 response element on p27) or5′-gacttgcatctagtcctgactccgg-3′ and 5′-gcctacctcatctcatacgctccag-3′(distal p27 element); and for beta-actin, 5′-aaactctccctcctcctcttcct-3′and 5′-cgagccataaaaggcaactttcg-3′. An equivalent volume ofunprecipitated genomic DNA was amplified as negative control.One-fifteenth (unprecipitated genomic DNA) or one-fifth (precipitatedDNA) of the PCR products were then separated on a 2% agarose gelcontaining ethidium bromide.

18. FRET

In order to evaluate the effect of a ligand on the recruitment ofTReP-132 by ER and PR, FRET (Fluorescence Resonance Energy Transfer) wascarried out between TReP-132 and PR or ER.

GST-LBD ER and GST-LBD PR fusion proteins (“GST” forGluthatione-S-Transferase and “LBD” for Ligand Binding Domain) wereexpressed and purified with the aid of GST-Trap columns (AmershamPharmacia).

The two NR-box peptides (nuclear receptor binding domain) of TReP-132(NH2-AVMDGAPDSALRQLLQKPMEPPAPA-COOH andNH2-FEAKGDVMVALEMLLLRKPVRLKCH-COOH) were synthesized and labelled withbiotin. GST-LBD ER, GST-LBD PR and NR-box peptides were respectivelydetected with AlloPhycoCyanin (APC) coupled with anti-GST antibody andwith R-phycoerythrin (RPE) coupled with streptavidin.

Increasing concentrations of compounds were incubated in binding buffer(0.1 M PBS, 2 mM CHAPS, 2 mM EDTA, 1 mM DTT, 0.1% BSA, pH 7.2) with 35nM GST-LBD ER or GST-LBD PR, 26.3 nM APC-labelled anti-GST antibody,1.25 nM RPE-strepavidin, 5-30 nM biotinylated NR-box peptide, at 4° C.for 4 hours in 384-well plates. The excitation wavelength for RPE was495 nm and emission was measured at 635 nm (RPE) or 670 nm (APC).Fluorescence intensities were measured on a Genesis Freedom 200 Tecan.The fluoresence ratios (intensity at 670 nm/intensity at 635 nm)relative to the ligand were calculated.

Results

1—Effect of TReP 132 on Cell Proliferation.

The effect of TReP-132 on cell proliferation was first demonstrated inHeLa cells by a colony formation assay. This experiment showed that HeLacells transfected with a pcDNA3-TReP expression vector formed far fewerG418-resistant colonies than cells transfected with pcDNA3 alone (FIG.11). 75 to 80% fewer colonies formed after transfection with thepcDNA3-TReP vector than after transfection with the control vector.Furthermore, when these cells were analyzed for expression of exogenousTReP-132, only two of 20 colonies were positive. When said two colonieswere dispersed and reseeded, the cell proliferation rate hadsignificantly decreased. The doubling time of cells expressing TReP-132was 30 hours versus 18 hours for control cells transfected with thepcDNA3 vector alone. Thus, these data show that the increase in TReP-132expression is correlated with the decrease in cell proliferation andtherefore is probably responsible for the smaller number of viablecolonies. To more clearly demonstrate that TReP-132 expression isclosely correlated with cell proliferation, human breast cancer MCF-7cells were treated for 6 days with 10 μM estradiol (E₂), a compoundknown to exert a proliferative effect on estrogen-dependent cells suchas breast cancer cells via induction of the G₁→S transition. Followingtreatment of MCF-7 cells with estradiol, RNase protection assaysrevealed a marked decrease in TReP-132 expression (FIG. 2) and aconcomitant increase in cell proliferation.

The effect of TReP-132 progesterone-dependent cell proliferation wasdemonstrated by measuring TReP-132 mRNA levels in T47-D cells treated ornot with progesterone. TReP-132 has previously been shown to be asteroidogenic factor expressed in T47-D breast cancer cells (Musgrove etal., 1997) expressing high levels of PR (Progesterone Receptor) (Mockuset al., 1982). The results in FIG. 3A show that TReP-132 mRNA levelsincreased with exposure time to progesterone, reaching a peak (3-fold)at 48 hours of exposure. These data thus show that TReP-132 expressionis increased by treating T47-D cells with progesterone.

The effect of TReP-132 on cell proliferation and response toprogesterone was then demonstrated by the Small Interfering RNA (siRNA)method. FIG. 3B shows that progesterone inhibited cell proliferationafter 4 to 6 days of treatment in transfected control cells, whereasTReP-132 knockdown led to an inhibition of cell proliferation, even inuntreated cells. These data indicate that TReP-132 is a cell growthinhibitory factor and that TReP-132 acts as a mediator of theanti-proliferative effect of progesterone.

In order to study the mechanism of action of TReP-132 in cell growthcontrol, clones with stable inducible expression of TReP-132 via theTet-off system were produced in HeLa cells (FIG. 4). In this manner,cell lines co-expressing GFP and TReP-132-Flag (HTO-GFP/TReP) or onlyexpressing GFP (HTO-GFP) were established. In this system, the cellswere cultured in the presence of doxycycline so as to inhibit expressionof TReP-132. The TReP-132-Flag protein was not detectable in cellscultured in the presence of Dox. On the other hand, when dox was removedfrom the medium, TReP-132-flag protein expression was detectable at 12hours and increased up to 48 hours (FIG. 5). Thus said cell lineconstitutes a suitable model to study the effects of TReP-132 expressionon cell proliferation. While HTO-GFP/TReP-132 cells cultured in thepresence of Dox resumed a normal cell rate with a doubling time of about16 hours, after 4 days of culture, removal of Dox considerably reducedcell proliferation to a doubling time of approximately 60 hours (FIG.6). Since no differences in cell proliferation were observed whenHTO-GFP cells and parent HTO cells were incubated with or without Dox,it can be concluded that overexpression of TReP-132 results in a stronginhibitory effect on HeLa cell proliferation.

2—Effect of TReP-132 on the Cell Cycle: Arrest in the G₁ Phase.

If TReP-132 is indeed involved in regulating cell cycle progression, itcan be expected that the expression thereof will also be regulatedduring the different phases of the cell cycle. To address this question,flow cytometry analysis of propidium iodide-stained cellular DNA showedthat induction of TReP-132 expression arrested cells in the G₁ phase(FIG. 7). In fact, starting from 48 hours after doxycycline removal,which corresponds to optimal induction of TReP-132 expression, anincrease in the proportion of cells in the G₁ phase and a concomitantdecrease in the proportion of cells in the S phase were observed.

In addition, HeLa cells were synchronized at the end of the G₁ phase bya double thymidine block. Twenty hours after the second incubation withthymidine, which corresponds to time 0 in these experiments, the cellswere harvested every 4 hours for cell cycle analysis by flow cytometryand for TReP-132 expression by RNase protection assays. TReP-132expression was induced at times T20 and T28, when the majority of cellswere in the G₁ phase (88 and 57% of cells, respectively) (FIG. 8). Incontrast, TReP-132 expression was not detectable when 82% of cells werein S phase, at time T24. This experiment shows that TReP-132 expressionis dependent on the G₁ phase of the cell cycle, which is in agreementwith the ability of said protein to arrest cell cycle progression inthis phase.

These data reveal an inverse correlation between TReP-132 expression andcell cycle progression.

3-TReP-132 Expression Leads to Inhibition of CDK Activities (CyclinDependent Kinases) and to a Decrease in Rb Phosphorylation(Retinoblastoma Protein).

Cell cycle progression is carried out by cyclin-dependent kinases (CDKs)which phosphorylate a number of key proteins involved in cell cyclecontrol (Schafer, 1998). As CDKs are constitutively expressed during thecell cycle, the regulation of their activity, which occurs only atspecific steps of the cell cycle, is ensured by post-transcriptionalmechanisms. For instance, CDKs are activated only by interaction withspecific cyclins, cyclins A, B, C, D1, or E, so named due to theircyclic expression during the cell cycle. Moreover, CDKs are inhibited byinteraction with CDK inhibitors (CDKIs), which compete for binding tocyclins and prevent their resultant activation. CDKI expression is alsoregulated during the cell cycle, as well as by growth factors (Owa etal., 2001), and in particular those of the insulin family (Stewart etal., 1990). It has also been shown that the respective levels of CDKIs(and cyclins) are altered by irradiating cells, thereby confirming theirinvolvement in processes associated with tumor development (Bernhard etal., 1995). The fact that TReP-132 was initially characterized as atranscription factor (Gizard et al., 2001) would suggest that itcontrols cell cycle progression by regulating the expression of proteinswhich are transiently induced during the cell cycle.

Progression through the cell cycle is controlled by a family of CDKswhose activity is regulated by phosphorylation, activated by binding tocyclins and inhibited by CDK inhibitors, which include the family of“CDK4 inhibitors” (“INK4”) (p16^(INK4A), p15^(INK4B), p18 and p19) andthe family of “protein kinase inhibitors” (“PKI”) (p21^(WAF/CP-1),p27^(KiP1) and p57^(Kip2)) (Sherr, 1994; Morgan, 1995; Sherr andRoberts, 1999). A key target of CDK action during the G₁ phase is theproduct of the retinoblastoma susceptibility gene (pRb) which induces G₁arrest by sequestering transcription factors from the E2F-DP family.Phosphorylation of pRb and other members of this family such as p107 andp130, through active cyclin-CDK complexes, leads to release of E2F andDP and to the resultant transcriptional regulation of target genesrequired to enter the S phase.

Phosphorylation of pRb and histone H1 by CDKs in the G₁ phase is a majorevent for progression of cells to the S phase (Peter and Herskowitz,1994; Zarkoska and Mittnacht, 1997). To determine whether TReP-132influences cyclin/CDK complex activities, the in vitro kinase activitiesof immunoprecipitated cyclin D1/CDK6 and cyclin A/CDK2 complexes weredetermined using pRb and histone H1 as substrate, respectively (Kitagawaet al., 1996; Koff et al., 1991). The results, presented in FIG. 9A,show that induction of TReP-132 expression in HTO-GFP/TReP-132 cells for48 hours reduced the in vitro kinase activities of the cyclin/CDKcomplexes immunoprecipitated in the G₁ phase (cyclin D1/CDK6 and cyclinA/CDK2).

Analyses performed directly on total protein extracts from HTO cells,shown in FIG. 9B, indicate that while pRb was mainly present in aninactive, hyperphosphorylated state (ppRb) in uninduced HTO cellsexpressing GFP and TReP-132 under control of doxycycline, TReP-132expression led to a significant increase in the amount ofhypophosphorylated pRb (pRb) and a marked reduction in phosphorylatedpRb (ppRb). Phosphorylated pRb levels decreased within 24 hours of Doxremoval and were almost undetectable at 72 hours, whereas no change inpRb phosphorylation was observed in HTO cells expressing GFP alone.These data therefore show that hypophosphorylation of pRb reached itsmaximum at 36 hours, a key time point preceding the maximum accumulationof cells in the G₁ phase (observed at 48 hours), thereby demonstratingthe role of TReP-132 in cell cycle arrest at the G₁ phase.

4-TReP-132 Upregulates the Expression of CDKIs (Cyclin Dependent KinaseInhibitors).

RNase protection assays were then carried out to determine whetherinduction of TReP-132 expression in HeLa cells was associated withchanges in the transcription of genes coding for the following CDKIs:p57, p27, p21, p19, p18, p16 and p27/p15, and that of factorscontrolling progression of the G₁ phase: Rb, p130, p107 and p53. Theresults showed that induction of TReP-132 expression by removal ofdoxycycline from the medium led to induction of the p27^(KiP1),p21^(CIP1/WAF1) and p16^(INK4) genes (FIG. 10). The fact that thesethree cyclin kinase inhibitors are involved in modulating G₁ progressioncorrelates with the fact that TReP-132 expression is induced and thatTReP-132 arrests the cell cycle at the G₁ phase.

The ability of TReP-132 to activate the promoters of these genes wasthen investigated. Increasing amounts of the pcDNA3-TReP-132 expressionvector were cotransfected with each reporter construct controlled by thepromoter regions of the p21, p16 and p27 genes. The p27 promoter showeddose-dependent activation by TReP-132, up to approximately 7-foldinduction, after cotransfection with 0.3 μg of the TReP-132 expressionvector (FIG. 11). The p21 and p16 promoters could also be activated to alesser extent by TReP-132 with a maximum 2 to 3-fold induction relativeto the control in the absence of TReP-132. These experiments clearlydemonstrate the direct role of TReP-132 in the transcription of key cellcycle regulatory molecules.

5. Characterization of this Activation with TReP-132 Acting as Mediatoron the p27 and p21 Promoters.

p21 and p27 are the CDKIs whose regulation has been most extensivelystudied, particularly in terms of their key role in the G₁→S transitionof the cell cycle. In fact, they inhibit the cyclin/CDK2 and cyclin/CDK4complexes whose activity is correlated with the onset of chromosomeduplication.

Transcription of p21 can be induced by progesterone. Owen et al. (1998)showed that said induction involves an interaction between theprogesterone receptor (PR), CBP/p300 and the Sp1 transcription factor,at the level of Sp1 DNA binding sites in proximity to the TATA box.Since TReP-132 interacts with CBP/p300 to increase transcription of theP450scc gene (Gizard et al., 2001), it was determined whether TReP-132induction of the p21 promoter similarly involved an interaction withCBP/300. To test this hypothesis, TReP-132, Sp1, or p300 expressionvectors were cotransfected separately or simultaneously. As previouslydescribed by Owen et al. (1998), Sp1 and p300 can induce the p21promoter and act additively when they are cotransfected simultaneously(FIG. 12). In the present invention, the inventors show that TReP-132has an additive effect on the activation conferred by Sp1 or p300, andthat maximum activation of the p21 promoter could be obtained when thethree transcription factors were co-expressed. The specificity of therole of p300 in said activation was demonstrated by co-expression of E1Awhich is known to have an inhibitory effect on p300 (Frangioni and Neel,1993; Monte et al., 1998). In the presence of E1A protein, thestimulatory effect was sharply reduced.

Previous studies have identified six proximal Sp1 binding sites in the−117/−51 region of the p21 promoter (Hong et al., 2002; Kardassis etal., 1999; Lu et al., 2000; Nakano et al., 1997; Pardali et al., 2000;Prowse et al., 1997; Santini et al., 2001; Somasundaram et al., 1997;Steger et al., 2002; Zhang et al., 2000) and two binding sites atpositions −544 and −536 of the p27 promoter (Lee et al., 2003; Ryhanenet al., 2003; Williamson et al., 2002) which play a major role in theregulation of these genes.

In order to study the mechanism of activation of the p21 and p27 genesby TReP-132, reporter constructs containing wt, mutant or deleted Sp1response elements were cotransfected with Sp1 and/or TReP-132 expressionvectors in HeLa cells. The results in FIGS. 13A and 13B show that, in asurprising manner, the activities of the −2320 p21 Luc and −3568 p27 Lucconstructs were induced in the presence of Sp1 or TReP-132 alone, thehighest activity being obtained when both factors were cotransfected.The results in FIG. 13B show that the shortest promoter construct (−154p21 Luc) containing the Sp1 response element was activated by Sp1 andTReP-132 but the response to Sp1 and/or TReP-132 was abolished when theSp1 response element was deleted. FIG. 13B shows that the −549 p27 Lucconstruct was still activated by Sp1 and/or TReP-132 whereas furtherdeletions in the region comprising the Sp1 sites abolished activation bySp1 or TReP-132, alone or together. Activation by Sp1 and TReP-132 wasabolished by mutation of nucleotide −544 in the −3568 p27 Luc construct.These data therefore indicate that TReP-132 and Sp1 interact at proximalSp1 binding sites of the p21 and p27 promoters.

Chromatin immunoprecipitation (ChIP) experiments were then performed toevaluate the interaction of TReP-132 and Sp1 with proximal Sp1 bindingsites in the p21 and p27 promoters in cells. To this end, theexperiments were carried out in HTO cells using an anti-Flag antibody toimmunoprecipitate the Flag-TReP-132 fusion protein. The results arepresented in FIG. 13C. The genomic DNA regions encompassing the Sp1response elements of the p21 and p27 promoters were immunoprecipitatedby the anti-Flag antibody in HTO cells expressing GFP and TReP-132 underdoxycycline control in which TReP-132 expression was induced, but not inHTO-GFP cells. PCR amplification using primers covering a regionapproximately 1 kb upstream of the Sp1 response elements of the p21 andp27 promoters or primers specific for the beta-actin gene did not resultin a significant signal, thus demonstrating the specificity ofimmunoprecipitation and PCR amplifications. These data show thatTReP-132 is part of a complex binding to proximal Sp1 binding sites inthe p21 and p27 promoters.

Gel shift assays using in vitro synthesized TReP-132 protein anddouble-stranded oligonucleotides encompassing the Sp1 sites on the p21and p27 promoters were then carried out to determine whether TReP-132directly binds to Sp1 elements. No DNA/protein complexes were detected,indicating that TReP-132 activates the promoter through interaction withDNA binding proteins other than Sp1. To test this hypothesis, pull-downassays were carried out with the GST or GST-TReP-132 fusion proteins andlabelled Sp1 protein. The results, in FIG. 13D, show that aprotein/protein itneraction was observed between Sp1 and theGST-TReP-132 fusion protein but no interaction was seen between Sp1 andthe GST protein.

6—Interaction of TReP-132 with Progesterone and Estrogen Receptors.

TReP-132 mediation of the cell growth inhibitory effect of progesteroneand the similar effects of TReP-132 and progesterone on theproliferation of cancer cells suggest that said two factors interact toregulate transcription of the p21 and p27 genes. Owen et al. (1998) hasshown that the p21 gene is activated by the bound progesterone receptor(PR) through interaction with Sp1 at the third and fourth Sp1 sites(respectively Sp1-3 and Sp1-4).

To test the interaction of TReP-132, PR and Sp1 at Sp1 binding sites onthe two promoters, reporter constructs containing the two promoters werecotransfected with the TReP-132 expression vector in T47-D cells and thecells were treated or not with progesterone. The results, presented inFIGS. 14A and 14B, show that TReP-132 activated the two promoters, asdid progesterone, whereas a higher level of activation was observed whencells were transfected with TReP-132 and also treated with progesterone.FIG. 14A shows that a mutation in the Sp1-3 site of the −93 p21 Lucconstruct completely abolished activation of the promoter by TReP-132and progesterone, alone or in combination, while mutation of the Sp1-4site reduced progesterone-dependent activation. Similarly, FIG. 14Bshows that deletion of Sp1 binding sites in the p27 promoter led to areduction of activation by TReP-132 and progesterone. These dataindicate that TReP-132 might cooperate with PR and Sp1 to mediate theeffects of progesterone on expression of the p21 and p27 genes. ChIPexperiments were then carried out to test this hypothesis in cells,using DNA from T47-D cells treated or not with progesterone andanti-TReP-132 antibody. The results in FIG. 14C show that the proximalSp1 response element of the p21 and p27 promoters was immunoprecipitatedin a complex obtained by using anti-TReP-132 antibody. TReP-132 bindingwas much more pronounced with DNA from progesterone-treated cells.Negative controls were DNA extracts amplified with primers covering aregion approximately 1 kb upstream of the Sp1 binding sites or primersspecific for the beta-actin gene for which no product has beenidentified.

Pull-down assays then showed that TReP-132 and PR interact (FIG. 14D,column 3) and that said interaction is increased in a dose-dependentmanner by progesterone (FIG. 14D, columns 3 to 7). TReP-132 contains twoNR-boxes (Nuclear Receptor box) and LXXLL motifs at amino acids 181(LRQLL) and 863 (LEMLL) (Gizard et al., 2001). Furthermore, it has beenshown that the presence of the amino-terminal LXXLL motif is essentialfor interaction with SF-1 (Gizard et al., 2002b). Pull-down assays werethen carried out with GST-TReP-132 proteins mutated at each LXXLL motif.FIG. 14D (right) shows that mutation of each motif led to a markedreduction in TReP-132 binding to PR, demonstrating the importance ofLXXLL motifs in the in vitro interaction of TReP-132 with PR. Takentogether, these data show that TReP-132 is a co-activator of PR in theregulatory complex formed with Sp1 at the proximal Sp1 binding site ofthe p21 and p27 promoters.

In the same manner, pull-down assays were carried out to test theinteraction of TReP-132 and ER. The results in FIG. 15 show thatTReP-132 and ER interact (line 3). From these data, together with theresults showing a marked decrease in TReP-132 expression associated withan increase in cell proliferation in MCF-7 cells treated with estradiol,it can be concluded that the action of ER on cell proliferation takesplace through a mechanism involving an interaction with TReP-132.

Additional FRET (Fluorescence Resonance Energy Transfer) studies alsodemonstrated the effect of a ligand on the recruitment of TReP-132 by ERand PR: the energy transfer between AlloPhycoCyanin (APC)-coupledanti-GST antibody to GST-PR or GST-ER fusion proteins, on the one hand,and streptavadin-coupled R-phycoerythrin (RPE) to the biotin/TReP-132NR-box complex on the other hand, demonstrated that binding betweenTreP-132 and the ER and PR receptors is ligand-dependent.

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1-12. (canceled)
 13. A method for the selection, identification,characterization or optimization of active compounds reducing cellproliferation, comprising determining the ability of a test compound tomimic, increase or promote the binding of the TReP-132 protein or of acomplex comprising the TReP-132 protein to the receptor of one or moresteroid hormones.
 14. The method according to claim 13, comprising a.contacting a test compound with: i. the TReP-132 protein or a complexcomprising the TReP-132 protein, and ii. the receptor of one or moresteroid hormones, and b. measuring the binding of the TReP-132 proteinor of the complex comprising the TReP-132 protein to said receptor. 15.The method according to claim 14, wherein the receptor is theprogesterone receptor or the estrogen receptor.
 16. A method for theselection, identification, characterization or optimization, in vitro orex vivo, of active compounds reducing cell proliferation, comprising: a.contacting, in the presence of progesterone or estrogens, a testcompound with: i. respectively, the progesterone receptor or theestrogen receptor, ii. Sp1, and iii. a nucleic acid comprising all orpart of the promoter of a target gene of progesterone or estrogens, andb. measuring the binding of Sp1 or of the complex comprising theprogesterone or estrogen receptor and Sp1 to said promoter, or measuringthe expression of said promoter, said measurement being an indication ofthe effect of the test compound on cell proliferation.
 17. The methodaccording to claim 16, wherein contact is carried out in the presence ofthe TReP-132 protein or a complex comprising the TReP-132 protein. 18.The method according to claim 16, wherein contact is carried out in acell.
 19. The method according to claim 16, wherein the target gene ofprogesterone or estrogens is selected in the group consisting of thep21, p16 and/or p27 genes.
 20. A method for the treatment of ahormone-dependent cancer by administering to a subject in need of suchtreatment a compound selected from among the TReP-132 protein, an analogof said protein, a compound increasing or mimicking the activity ofTReP-132 and a compound obtained with the aid of a method according toclaim 13, in combination with one or more steroid hormones in view of asimultaneous, separate or sequential administration.
 21. The methodaccording to claim 20, wherein the hormone is progesterone or anestrogen.
 22. The method according to claim 20, whereinhormone-dependent cancer is breast cancer or uterine cancer.
 23. Apharmaceutical composition, comprising, in a pharmaceutically acceptableexcipient, a compound selected from among the TReP-132 protein, ananalog of said protein, a compound increasing or mimicking the activityof TReP-132 and a compound identified with the aid of a method accordingto claim 13, combined with one or more steroid hormones, in view of asimultaneous, separate or sequential administration for treating ahormone-dependent cancer.
 24. The pharmaceutical composition accordingto claim 23, wherein the hormone is progesterone or an estrogen.